Conformational and topological protein regulation

ABSTRACT

Methods and compositions are provided for identifying novel conformers of proteins not known to exist as different conformers. By using chimeric genes replacing a native signal sequence with a different signal sequence resulting in the production of a conformer, one can compare the native protein with the product of the chimeric gene. Where the conformations are different, the different protein may be used for production of antibodies, elucidation of mechanisms associated with the native and different conformer protein, assays for the presence of the different conformer in physiological samples, identification of compounds specifically binding to the conformer, particularly drugs, etc. Where the formation of the conformer is associated with a diseased state, the conformer may be used in screens to identify compounds as drug candidates.

CROSS REFERENCE TO RELATED APPLICATIONS

This specification claims the benefit of U.S. Ser. No. 60/171,012 filedDec. 15, 1999 and U.S. Ser. No. 60/172,350 filed Dec. 16, 1999, whichdisclosures are incorporated herein by reference.

INTRODUCTION

1. Technical Field

The field of this invention is biological models and therapeutics.

2. Background

Conventionally, gene expression in higher eukaryotes is generallybelieved to involve the conversion of information encoded in the genomeinto proteins through the processes of transcription, RNA splicing andexport, and translation. Localization of newly synthesized proteins tothe correct intracellular compartment, and folding into the correcttertiary structure, are two additional processes at which geneexpression could, in principle, be regulated. However, until now theformer has been viewed as regulated only with respect to fidelity (Songet al, Cell (2000) 100:333-343), degradation (Wiertz et al, Nature(1996) 384:432-438), or signaling to other compartments (Sidrauski etal., Trends Cell Biol (1998) 8:245-249), and not with respect to theinformation content of the expressed gene. Likewise, folding has alsonot been viewed at a point of fundamental regulation of gene expression.In general, a dichotomy has been accepted between a protein being eitherproperly folded or misfolded (Ellgaard, et al., Science (1999)286:1882-1888). The possibility that proteins might have more than oneproperly folded state and that the cell might be able to select oneversus another folded state under particular circumstances orconditions, has not been seriously considered. If either of thesepossibilities were true, the information content of the genome would begreatly increased. If an invention made it possible to select oneconformation versus another, such an invention would be a platform fordetermining and accessing the information content of the genome in waysthat have not been heretofore possible.

In regards to protein folding, a fundamental dogma of modern biology isthat primary structure determines secondary structure, which, togetherwith relevant post-translational modifications such as glycosylation,determines the tertiary structure of proteins (Anfinsen, Science (1973)181:223-230). One revision of this view occurred with the realizationthat molecular chaperones play a crucial role in enhancing the fidelityof protein folding by preventing inappropriate interactions, therebyfacilitating the process of achieving the proper final folded state(Ellis and Hartl, Faseb J (1996) 10:20-26). The recognition that foldingis likely initiated in many parts of the molecule at the same time,allowing the chain to funnel towards a minimum energy state withoutsampling every possibility along the way, constituted a second revisionin the generally accepted view of protein folding (Dill and Chan, NatureStruct Biol (1997) 4:10). Neither of these notions considers thepossibility that folding might be regulated in the sense of proceedingdown one versus another pathway contingent on one versus another set ofprotein-protein interactions. If this were the case, protein foldingcould be amenable to manipulation in ways that could confer diagnosticor therapeutic advantage.

Proteins destined to be secreted from the cell generally contain asignal sequence at the amino terminus that initiates a series ofprotein-protein interactions directing the growing chain to theendoplasmic reticulum (ER) membrane and through the translocationchannel into the ER lumen (Blobel, PNAS (1980) 77:1496-1500). The rolesof signal recognition particle and its receptor (Walter and Johnson,1996) and of the heterotrimeric Sec 61 complex (Gorlich and Rapoport,Cell (1993) 75:615-630) and of other proteins (Jungnickel and Rapoport,Cell (1996) 82:261-270) in these processes has been elucidated.

Assembly of integral membrane proteins into the membrane of the ERappears to be a complex variation on this general theme, directed byinternal signal sequences and stop transfer sequences and hybridsignal-anchor sequences whose interaction with various ER proteinsdirects the final transmembrane orientation of the polypeptide and oftenplay a subsequent role in anchoring the protein in the bilayer after thechain is released from the translocation channel into the lipid bilayer(Skach and Lingappa, J Biol Chem (1993) 268:23552-23561; Borel andSimon, Cell (1996) 85:379-389; Heinrich et al, Cell (2000) 102:233-244;Moss et al., Mol Biol Cell (1998) 9:2681-2697).

In the case of secretory proteins, the signal sequence is usuallycleaved from the growing chain by the ER membrane associated signalpeptidase complex, trapping the nascent chain in the ER lumen (Matlacket al., J Cell Biol (1999) 270:6170-6180), with the cleaved signalpeptide most likely returned to the cytosol and degraded (Lyko et al., JBiol Chem (1995) 270:19873-19878). In bacteria and primitive eukaryotessuch as yeast, a substantial amount of translocation occurs aftersynthesis is completed (Rapoport et al., J Biol Chem (1999)380:1143-1150). In such post-translational translocation, a role for thesignal sequence as a molecular chaperone to maintain the unfolded statehas been proposed (Liu et al., PNAS (1986) 86:9213-9217). However inhigher eukaryotes, where most translocation across the ER membraneappears to occur co-translationally, that is, while the chain is stillbeing made, it has generally been assumed that the nascent chain istransferred directly to the ER lumen where folding is initiated (Chenand Helenius, Mol Biol Cell (2000) 11:765-772).

Taken together, the studies presented here suggest the need for severalrevisions in the current paradigm of protein folding:

First, the simple dichotomy between properly folded and misfoldedproteins must be abandoned. It needs to be recognized that proteinfolding can result in multiple properly folded states which may,potentially, subserve different functions. In most cases these areextremely difficult to detect not only because tools to easilydistinguish conformational variants of proteins are limited and noteasily applied, but also because the cell complicates the taskby-degrading variants not wanted at a given point in time.

Second, it must be recognized that the cell has mechanisms by which onefolded state (and therefore one function) is chosen (to survivedegradative mechanism and be exported to the surface or out of the cellat one time while another folded state (and another function) may bechosen at another time.

Third, it is clear that the machinery and determinants involved intranslocation across the ER membrane play an important role in selectingthe folding funnel down which a newly synthesized protein proceeds, andthat manipulation of either the signal sequence or the machinery withwhich the chain interacts in the cytosol, membrane or ER lumen are waysto change the folding funnel selected, and therefore, the finalconformation or mix of conformations of the protein.

The major implication of the first two revisions of the protein foldingparadigm is to increase the information content of the genomeenormously. The major implication of the third is to reveal a means ofaccessing this increased information content of the genome fordiagnostic and therapeutic advantage, giving rise to the subjectinvention.

Besides elucidating these points of background, our studies demonstratean invention by which the choice of folding funnel can be altered forany given protein in a way that does not change the proteins finalprimary structure, but which can result in dramatic changes in theprotein's final folded conformation or shape. We demonstrate the signalsequence is able to influence the folding funnel utilized by the growingpolypeptide chain, and thereby, to influence the final foldedconformation of the resulting newly synthesized protein. Furthermore,cells are able to choose one versus another final conformation throughsignal sequence-mediated regulation. Because a linear polypeptidesequence folding in different ways could have substantially differentshapes, physical properties and biological activities, this new level ofregulation greatly increases the information content of the genome andthe potential for regulation of gene expression available to the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects on topology of replacing the PrP signalsequence. FIG. 1(A) shows schematic diagrams of wild-type PrP, Prl-PrP,and βL-PrP. Boxes represent signal sequences from PrP, preprolactin, andpre-beta-lactamase, fused to the PrP mature region. Cleavage site isindicated by arrowhead. FIG. 2(B) shows the topology of PrP, Prl-PrP,and βL-PrP. Microsomes from in vitro translation and translocationreactions of each construct were isolated by sedimentation and dividedinto equal aliquots for digestion with proteinase K (PK) in the presenceor absence of 1% Triton X-100 (detergent) as indicated below the gel.One aliquot was left untreated. The three topological forms of PrP yielddistinctively sized bands upon digestion with PK in the absence ofdetergent (Hedge et al. (1988a) Science 279, 827-834). The position ofeach band, along with a diagrammatic representation of the respectivetopology, is provided to the left of the gel. Cleavage of the signalsequence of Prl-PrP was inefficient, causing ^(sec)PrP and ^(Ntm)PrP tobe represented by both a signal-cleaved species and a signal-uncleavedspecies. Molecular weight markers are at right. The percentage oftranslocated chains in the ^(Ctm)PrP topology for each construct wasquantitated from 3 such experiments and is shown in (C) (mean+/−SEM).

FIG. 2 shows that kinetics of targeting do not influence PrP topology.FIG. 2 (A) shows PrP translated in the presence of varying amounts ofmicrosomes as indicated on the x-axis. Following proteolysis assays fortopology, autoradiographs were quantitated to determine the percentageof total translation product that was signal cleaved (closed squares),and the percentage of translocated chains in the ^(Ctm)PrP topology(open circles). FIG. 2(B) shows translation reactions of PrPsynchronized by inhibition of initiation at 5 minutes with 75 mM ATA. Atstaggered intervals (as indicated on the x-axis), aliquots were removed,microsomes were added, and targeting and translocation were allowed toproceed for 30 minutes. Topology and signal cleavage were determined andplotted as in FIG. 2(A). FIG. 2(C) shows an StuI-truncated mRNA,encoding the 223-mer of PrP, translated in the presence (open bars) orabsence (shaded bars) of microsomes. After 5 minutes, further initiationwas inhibited with ATA, and at 30 minutes, microsomes were added to thesample lacking them and the incubation for both reactions was continuedfor an additional 30 minutes. Nascent chains were then released from theribosomes with 10 mM EDTA. Signal cleavage and topology were assessedand quantitated as above.

FIG. 3 shows that the signal sequence influences the ribosome-membranejunction. FIG. 3(A): Truncated mRNAs for preprolactin (Prl),pre-beta-lactamase (βL), βL-Prl and Prl-βL were translated andtranslocated, and the microsomes from these reactions were isolated bysedimentation. One aliquot of each sample was left untreated and PK wasadded to the remainder. Prl and βL-Prl were truncated at PvuII. βL andPrl-βL were truncated at HpaII. The sizes of these constructs in aminoacids are indicated. FIG. 3(B): Prl-PrP, wild-type PrP, and βL-PrP weretruncated at BstXI (61 aa for wild-type PrP), NaeI (113 aa), XbaI (135aa), or HincII (180 aa) and analyzed by proteolysis as in FIG. 3(A). Thepercentage of material protected from digestion by PK was measured bydensitormetric quantitation of the autoradiographs shown, and is givenbelow each sample. The drawings at right represent the position of boththe signal sequence (open box) and transmembrane domain (gray box) withrespect to the ribosome.

FIG. 4 shows dissociation of topology from TRAM dependence. FIG. 4(A):signal sequences of constructs PrP, PrP_((R2,3)), PrP_((D2,3)),PrP_((R4,5)), and PrP_((D4,5)). The amino acid changes in each constructare underlined, and the site of signal cleavage is indicated by anarrowhead. FIG. 4(B): the constructs shown in FIG. 4(A) were used toprogram translation and translocation reactions, and the resultanttopology analyzed by proteolysis as in FIG. 1B. The percent of ^(Ctm)PrPsynthesis was quantitated by densitometry of the autoradiographs and isshown for each construct. The signal sequence of each construct isindicated on the x-axis. ^(Ctm)PrP synthesis for Prl-PrP and βL-PrP isshown for comparison. FIG. 4(C): the PrP signal sequence mutations shownin FIG. 5A were fused to the prolactin mature region. The signalsequences of prepro-alpha factor (dependent) and preprolactin(independent), fused to the prolactin mature region, served as controlsfor TRAM-dependent and TRAM-independent signal sequences, respectively(see Voigt et al., (1996) J. Cell. Biol. 134, 25-35). Constructs weretranslated and translocated in ConA-depleted proteoliposomes containingor lacking purified TRAM (prepared as described in the ExperimentalProcedures). An aliquot from each reaction was set aside to verify thatthe efficiency of translation was identical in both types ofproteoliposomes. The remainder of the material was treated with 0.5mg/mr PK and immunoprecipitated with anti-protein antibodies. TRAMdependence, shown here, was measured as the percent diminishment ofprotease protected material in the proteoliposomes lacking TRAM relativeto TRAM-containing membranes.

FIG. 5 shows relationship between the signal sequence and TM domain.FIG. 5(A): Constructs PrP_((AV3)), Prl-PrP_((AV3)), andβL-PrP_((AV3))were translated, translocated, and analyzed for topologyby proteolysis exactly as in FIG. 1B. The migration of each topologicalform is indicated at right. FIG. 5(B): Constructs PrP_((G123P)),Prl-PrP_((G123P)), and βL-PrP(G123P) were translated, translocated, andanalyzed for topology by PK exactly as in FIG. 1B. Only ^(sec)PrP isobserved, and its migration is indicated at right. FIG. 5(C):HincII-truncated mRNAs coding for ˜180-mers of PrP, Prl-PrP_((AV3)). andβL-PrP_((G123P)) were translated in the presence of microsomes, whichwere then isolated by sedimentation. Proteolysis with PK to examine thestate of the ribosome-membrane junction was performed exactly as in FIG.3B.

FIG. 6 shows the interaction of ^(SBC)PrP with PDI. FIG. 6(A):HincII-truncated mRNAs coding for PrP_((G123P)) and PrP_((AV3))(˜180-mers) were translated, and the membranes were isolated bysedimentation. One aliquot was treated with 1 mM DSS (+XL) and theremainder was left untreated (−XL). Uncrosslinked material is shown atthe bottom of the gel. The migrations of the molecular weight markersare shown at right. The arrowhead indicates a protein of approximately65 kDa which cross-links preferentially to PrP_((G123P)). FIG. 6(B):Prl-PrP and βL-PrP were truncated at HincII and treated exactly as inFIG. 6(A). Equal amounts of total translation product were loaded ineach lane. The 65 kDa cross-link seen in (A) is indicated by anarrowhead. FIG. 6(C): Prl-PrP and βL-PrP were truncated at NaeI(˜113-mers) and analyzed by cross-linking exactly as in FIG.(A). The 65kDa cross-link seen in FIG. 6(A) (closed arrowhead) and a 60 kDacross-link which also preferentially forms with Prl-PrP (open arrowhead)are indicated. FIG. 6(D): Purification of PDIp. Shown is a Coomassiestain of starting rough microsomes (RMs); proteins not extracted(pellet) or extracted by saponin treatment; flow-through (FT) and eluateof the saponin extract from a ConA-sepharose column; flow-through andeluate of the ConA eluate from a Q-sepharose column, and the pooled peakfractions from a gel-filtration (GF) column, resulting in purificationof p65 (arrow) to near-homogeneity. See Experimental Procedures fordetails of the purification. FIG. 6(E): Prl-PrP and β-PrP weretranslated in the presence of either pancreas (top panel) or brain(bottom panel) microsomes, and the membranes were isolated bysedimentation. Following cross-linking with 1 mM DSS, the nascent chainswere released from the ribosomes with 10 mM EDTA, and 0.5% saponin wasadded to extract the lumenal proteins. One aliquot was set aside (lanes1, 4), and antibodies against PDIp (lanes 2, 5) or PDI (lanes 3, 6) wereadded directly to the remaining material for immunoprecipitation.Prl-PrP translated in pancreatic microsomes cross-links preferentiallyto PDIp (closed arrowhead) and PDI (open arrowhead), while only across-link to PDI (open arrowhead) is observed in brain microsomes. FIG.6(F): mRNAs encoding wild-type PrP and signal sequence point mutantsfrom FIG. 4A, truncated at Nael to yield ˜113-mers, as well as wild-typepreprolactin (Prl) truncated at MboII to yield a 100-mer, weretranslated and analyzed by cross-linking as in panel (A). Followingcross-linking and nascent chain release with 10 mM EDTA, 0.5% saponinwas added, the membranes were sedimented, and the supernatant,containing lumenal proteins, was recovered. Arrowheads designate^(sec)PrP-specific lumenal cross-links. The signal sequence of eachconstruct is indicated below the gel.

FIG. 7 shows a hypothetical model for signal sequence action. The signalsequence determines the state of the ribosome-membrane junction, whichin turn influences final topological fate in the case of PrP, andperhaps more subtle aspects of translocational behavior or othersubstrates. An example of each translocational outcome is given.

FIG. 8 shows a dose response of ^(Ctm)PrP induced neurodegeneration.FIG. 8(A): Topology of wild type and mutant PrP molecules at the ER. Invitro synthesized transcript coding for each PrP construct (indicatedabove the gels) was used to program a rabbit reticulocyte lysatecell-free translation reaction containing ER derived microsomalmembranes and a competitive peptide inhibitor of glycosylation.Following translation, samples were either left untreated or digestedwith PK in the absence or presence of 0.5% Triton X-100 (“Det”) asindicated above the gel. The positions of the full-length PrP molecule,the NH₂-terminal fragment (indicative of ^(Ntm)PrP) and theCOOH-terminal fragment (indicative of ^(Ctm)PrP) generated by PKdigestion are marked at the right. FIG. 8(B): Level of expression ofvarious transgenic mouse lines. Varying amounts in (μl, indicated abovethe lanes) of 10% brain homogenate from each transgenic mouse wasimmunoblotted for PrP with 13A5 monoclonal antibody and compared to atitration of syrian hamster brain homogenate. FIG. 8(C): Time course ofdevelopment of symptoms in Tg[SHaPrP(A117V)_(H)] (closed circles) andTg[SHaPrP(N108I)_(H)] (open circles). Non-transgenic control animals(dashed line) and Tg[SHaPrP(KH→II)_(H)] (closed squares; data fromManson, et al. (1994) Neurology 46:532-537) are shown for comparison.FIG. 8(D): Analysis of various transgenic mice and Syrian hamster (asindicated above the gel) for ^(Ctm)PrP and Prp^(Sc) as described inMethods. Tg[SHaPrP(A117V)_(H)] and Tg[SHaPrP(N108I)_(H)] samples werefrom clinically ill mice and Tg[SHaPrP(A117V)_(L)] andTg[SHaPrP(N108I)_(L)] samples were from mice (which showed no signs ofdisease) at least 600 days of age. The fragment of PrP resistant to PKunder the ‘mild’ conditions, indicative of ^(Ctm)PrP is only seen in theTg[SHaPrP(A117V)_(H)] and Tg[SHaPrP(N108I)_(H)] samples. No PK resistantPrP^(Sc) was observed in any of the samples.

FIG. 9 shows a relationship between ^(Ctm)PrP and PrP^(Sc). FIG. 9(A),(C), (E): Time course of development of illness in various transgeniclines following inoculation with Sc237 hamster prions. FIG. 9(B), (D),(F): Relative levels of protease-resistant Prp^(Sc) at time of illnessin various transgenic lines. Duplicate samples of each line weredigested using ‘harsh PK’ conditions as described in Methods andequivalent amounts of each sample separated by SDS-PAGE. The C-terminalPrP27-30 fragment characteristic of PrP^(Sc) (indicated with a bracket)was detected by immunoblotting with the 13A5 monoclonal antibody. Theposition of undigested, full length PrP is indicated with an asterisk.FIG. 9(G): The Ctm-index for each transgenic line (see Table 1) wasplotted against the amount of PrP^(Sc) accumulated at the time ofillness following inoculation with Sc237 prions.

FIG. 10 shows a lack of transmission of CtmPrP induced neurodegenerativedisease. Terminally ill Tg[SHaPrP(KH→II))_(H)] mice (‘KH→II’) andclinically normal Tg[SHaPrP] mice (‘wt’) were sacrificed and homogenatesof the brain tissue inoculated intracerebrally into various hosts asindicated below the graph. The host animals used wereTg[SHaPrP(KH→II)_(L)] [‘(KH→II)_(L)’], FVB/Prnp^(0/0) (‘PrP null’),Tg[SHaPrP], and Syrian hamsters. The time (in days) at which individualanimals died following inoculation is plotted. Deaths by all causes,including those related to the inoculation itself, are plotted. Theexperiments were terminated after ˜600 days with none of the remaininganimals showing any signs or symptoms of neurologic disease. Eachexperiment represents three sets of 10 host animals injected withinoculi prepared from three separate animals to be tested. It should benoted that in our animal care facility, hamsters routinely liveapproximately 200 to 400 days, while mice last greater than 600 days(data not shown).

FIG. 11 shows ^(Ctm)PrP generation during time course of PrP^(Sc)accumulation. FIG. 11(A): Schematic of experimental design. Doubletransgenic mice expressing both SHaPrP and MoPrP (represented by theshaded and open circles, respectively) are inoculated with RML mouseprions (crosshatched squares). Over time, host MoPrP^(C) is converted toMoPrP^(Sc) and accumulates. During this time course, the HaPrP is notconverted to HaPrP^(Sc) owing to the species barrier and may thereforebe assayed for ^(Ctm)PrP. FIG. 11(B): Relative amounts of total PrP^(Sc)and SHaPrP^(Sc) in mice at various times (in weeks) after inoculationwith RML. Homogenate was digested using the ‘harsh PK’ conditions,treated with PNGase, and analyzed by SDS-PAGE and immunoblotting witheither R073 polyclonal antibody (to detect total PrP) or the 3F4monoclonal antibody (to selectively probe for SHaPrP). An equivalentamount of homogenate is analyzed in each lane except lanes 2 and 11(which contain one-fourth as much) and lanes 3 and 10 (which containone-tenth as much). FIG. 11(C): Relative amounts of hamster ^(Ctm)PrP(detected selectively using the 3F4 monoclonal antibody) at varioustimes after inoculation with RML. Each bar represents the average ±SEMof three determinations.

FIG. 12 shows a three stage model of prion disease pathogenesis. Stage Iis the formation and accumulation of PrP^(Sc). This could be initiatedby either inoculation or spontaneous conversion of a mutated PrP^(C) toPrP^(Sc). Stage II comprises the events involved in generating^(Ctm)PrP. These events could be affected in trans at a presentlyunknown step (dashed lines with question marks) by accumulated PrP^(Sc)or in cis by certain mutations within PrP. Stage III represents theevents (currently unknown) involved in ^(Ctm)PrP mediatedneurodegeneration. This likely involves exit of ^(Ctm)PrP to a post-ERcompartment as a first step. In this model, PrP^(Sc) can cause disease(via ^(Ctm)PrP), but is not absolutely necessary, whereas ^(Ctm)PrPproduction is necessary and sufficient for the development of disease.

FIG. 13 shows efficiency of model secretory protein signal sequences.Full-length mRNAs for preprolactin (Prl), pre-beta-lactamase (βlac), andpre-IgG heavy chain (IgG) were translated in a rabbit reticulocytelysate in the absence or presence of canine pancreatic microsomalmembranes (RM), and equal aliquots of the translated material were leftuntreated or treated with Proteinase K (PK) in the presence or absenceof 1% Triton X-100 (det). The positions of unprocessed material (pPrL,pβlac, pIgG) are indicated, as are the positions of signal-cleaved (Prl,βlac) and glycosylated (IgG-CHO) material.

FIG. 14 shows that nascent secretory proteins act differentially on theribosome-membrane junction. FIG. 14(A): Preprolactin mRNA was truncatedat successive locations giving rise to nascent chains containing thenoted number of N-terminal amino acids. The number of amino acids of thesignal-cleaved protein is given in parentheses. The state of theribosome-membrane junction was assessed by proteolysis exactly as inRutkowski et al. (“A New Role for the Signal Sequence in TranslocationalRegulation”, see priority applications 60/171,012 and 60/172,350) FIG.14(B) and (C): βlac and IgG mRNAs were serially truncated at theindicated locations and analyzed by proteolysis as in (A). For eachpanel the cartoon to the right depicts the state of theribosome-membrane junction at each of the three points during chaingrowth. Note that the left panel only of FIG. 14A and the left andcenter panels of B and C depict an “open” ribosome-membrane junction,while the center and right hand panels of FIG. 14A and the right handpanels of FIG. 14B and C depict a “closed” ribosome-membrane junction.

FIG. 15: Prolactin (Prl) and IgG/Prl were truncated at PvuII (encodingthe signal sequence and 56 amino acids of the mature region of eachprotein) and translated in the presences of canine pancreatic roughmicrosomes. Targeted chains were isolated by sedimentation. One aliquotwas set aside and the other was treated with 2 mM BM(PEO)3 [XL]. Thearrowhead indicates a protein of approximately 35 kDa which cross-linkspreferentially to Prl. 10 kDa uncross-linked material is at the bottomof the panel. Migration of molecular weight markers is indicated to theleft of the gel.

FIG. 16: Glycosylation as a reporter of signal sequence-dependentconformational change. FIG. 16(A): in vitro-transcribed mRNAs encodingnative preprolactin (Prl) with an engineered N-terminal glycosylationsite, or glycoprolactin fused to the signal sequences of mouse IgG heavychain (IgG/Prl), rat growth hormone (GH/Prl), or hamster PrP (PrP/Prl),were translated in a rabbit reticulocyte lysate system, either in thepresence or absence of canine rough microsomes (RM) as indicated. Acompetitive inhibitor of glycosylation was also added to some samples(AP). Following translation, samples were either set aside or incubatedwith 0.5 mg/ml proteinase K (PK) in the presence or absence of 1% TritonX-100 (det) at 0° for 30 minutes. The reactions were then run onSDS-PAGE and visualized by autoradiography. Three species of prolactinare indicated: signal-cleaved, translocated, glycosylated prolactin(Prl-CHO); signal-uncleaved untranslocated preprolactin (pPrl); andsignal cleaved, translocated, nonglycosylated prolactin (Prl). FIG.16(B): The percentage of prolactin chains achieving glycosylation foreach construct was quantitated from three experiments by scanning anddensitometric analysis of the autoradiographs such as those shown in(FIG. 16(A). FIG. 16(C): COS-1 cells were transiently transfected withexpression plasmids encoding glycoprolactin with its own signal sequenceor those of rat growth hormone or mouse IgG heavy chain. For comparison,the same constructs lacking glycosylation sites were also transfected(-CHO). Following a 45 minute preincubation of the cells in serum-freemethionine-free medium, 100 μCi of a ³⁵S-Methione/Cysteine mixture wasadded to the cell medium and the cells were labeled for 1 hour. Analiquot of the media from these cells was run directly on SDS-PAGE. Bothglycosylated and unglycosylated forms of prolactin are indicated to theright of the panel. While glycosylated GH/Prl and IgG/Prl are clearlyvisible, no glycosylated native prolactin is observed.

FIG. 17 shows requirement of junctional regulation for successfultranslocation. FIG. 17(A): The prolactin mature region was fused toeither its own signal sequence (Prl-Prl) or those of pre-beta-lactamase(βL-Prl) or pre-IgG heavy chain (IgG-Prl) at the site of signal sequencecleavage. Full-length mRNAs for these substrates were translated in thepresence of microsomal membranes and analyzed as in FIG. 17A. For eachsubstrate, the efficiency of translocation was determined byquantitating the percentage of total chains which achieved a processed,translocated state. Each bar represents the mean percent translocationfrom three trials (+/−SEM). FIG. 17(B): mRNAs encoding Prl-Prl, βL-Prl,and IgG-Prl were truncated at either Pvull or AflII. The number of aminoacids from mature prolactin is shown in parentheses. Translated,sedimented chains were analyzed by proteolysis as in FIG. 15B to assessthe state of the ribosome-membrane junction. The y-axis plots the meanpercent protection of each substrate from PK in three trials. FIG.17(C): Schematic of IgG-Prl chimeras. Increasing lengths of pre-IgGheavy chain or a non-IgG stretch of amino acids were fused to theprolactin mature region. The site of signal sequence cleavage is shownby an arrowhead. Sequences from IgG are represented by open boxes, Prlmature region sequences by bars, and the non-IgG control sequence by ahatched box. The Prl signal sequence is indicated by a shaded box.Numbers below each construct represent amino acids from pre-IgG heavychain, the preprolactin signal sequence (Prl-Prl), or the non-IgGsequence (numbers 15-132 of IgG-Prl(1-132)Stuffer) with the initiationmethionine as number 1. FIG. 17(D): The substrates shown in FIG. 17(C)were translated in the presence of microsomal membranes and analyzed forthe mean percent translocation from three trials as in FIG. 17(B).

FIG. 18 shows a schematic of how translocational regulation can lead toconformational heterogeneity. Indicated in Roman numerals to the leftare the endpoints of three stages of protein biogenesis at the ER: I,the earliest events including targeting; II, the events of translocationper se; and III, the final folded protein. Translocational regulation,of which four forms are indicated as FIG. 18(A-D) in stage II, providesthe means by which heterogeneity is achieved among completed, foldedproteins (see III), as hypothesized here. Molecular chaperones areindicated by solid ovals, while TrAFs are depicted as hatchedrectangles. In FIG. 18(A), the translocon serves as a molecularchaperone. In FIG. 18(B), the translocon forces the nascent chain toinitiate folding in a reducing environment, perhaps in association withmolecular chaperones or machinery for post-translational modifications.Note that the lumenal gate of the translocon is closed, while that onthe cytoplasmic side that makes up the ribosome-membrane junction, isopen. In FIG. 18(C), the converse is achieved—a closed cytoplasmic gateand an open lumenal gate, again with the implicit participation ofdistinct molecular chaperones. Finally, FIG. 18(D) indicates that theaction of TrAFs can result in a change in protein topology as well asconformation. First order protein folding is independent of theseconsiderations. Second order protein folding manifests itself indifferent ways (note different molecular chaperones in FIG. 18B vs C)depending on TrAF action which constitutes the third order level ofcomplexity of protein folding. The final order of complexity of proteinfolding is due to the regulation of TraAFs by signaling pathways thatallow cells to choose between Figures (A-D), for example, in response toa change in environmental or other conditions.

FIG. 19: PrP cDNA was transcribed and translated as described previously(Lopez et al, Science (1990 248:226-229) in the presence of cytosolicextract prepared from mouse erythroleukemia cells before and 4 of 6 daysafter induction of differentiation with dimethylsulfoxide. Thee presenceof 35S methionine during the translation reactions allows radiolabellednewly synthesized proteins to be visualized by polyacrylamide gelelectrophoresis in sodium dodecyl sulfate (SDS-PAGE) and autoradiography(AR). One unit of activity is the amount of extract needed to change theratio of PrP topological forms in favor of sec PrP in a 10 μltranslation reaction supplemented with dog pancreas microsomal membranesat a final concentration of 5 A280 u/ml. To prepare the cytosolicextracts, cells were dounce homogenized after swelling in 10 mM Hepes pH7.5 and membranes removed by centrifugation at 100,000×g for 1 hr. Ascan be seen, cytosols from undifferentiated MEL cells are rich in anactivity that promotes secPrP, and that activity is lost upondifferentiation of the cells, over time. In this case PrP is serving asa reporter of cytosolic regulatory activities that are likely utilizedby several proteins for different endpoints. Note the presence of aglobin band in the gel from the 6 day point, indicating induction ofglobin, a differentiation marker in these cells.

FIG. 20: Transcription and translation of PrP cDNA was performed aspreviously described in the presence of microsomal membranes from theindicated sources. The products were subjected to protease digestion inthe absence or presence of detergent as previously described. Shown arethe products of digestion with protease in the absence of detergent,allowing the ratio of Sec-PrP to Ctm-Prp to be readily assessed bySDS-PAGE and AR. E=microsomal membranes from hamster brain on embryonicday 13; N=neonatal hamster brain microsomal membranes; 7d=microsomalmembranes from 7 day post natal hamster brain; 14d=microsomal membranesfrom 14 day post natal hamster brain; 21d=microsomal membranes from 21day post natal hamster brain; A=microsomal membranes from adult hamsterbrain; SUE=sea urchin embryo microsomal membranes (which lack TrAF andtherefore generate exclusively Ctm-PrP comparable toglycoprotein-depleted fractionated and reconstituted proteoliposomes(Hegde et al., (1998) Mol Cell 2:85-91). Upper arrow indicates theposition of SecPrP; lower arrow of Ctm-PrP. Graph below isquantification of the ratio of secretory PrP to total PrP for each ofthe indicated lanes above. What is demonstrated is that membranes fromdifferent tissues and species, and from the same tissue and species atdifferent times in development, can have radically different TrAFactivities, consistent with the hypothesis that translocation is animportant site of regulation of protein biogenesis, and thattrans-acting factors contribute to translocational regulation. In thiscase, prion protein is being used solely as a reporter of TrAF activity.The same principle applies to all genes for which translocationalregulation may be found.

SUMMARY OF THE INVENTION

Methods and compositions are provided for producing proteins of varyingtopography and/or topological species, providing chimeric proteins,identifying specific agents involved with the formation of topographicaland/or topological species, model in vitro and in vivo systems, andmethods for identifying topographical and/or topological distinctproteins. Proteins of varying conformation are produced by varying thesignal sequence, replacing the wild-type signal sequence with sequencesof known mechanism, selectively including translocon associated proteinsin an in vitro translocation model system, employing modified lysateswith microsomes for investigating and producing conformationallydistinct proteins, for producing and employing knock-out and mutantsmall laboratory mammals resulting in modulation of the topographicalproduction of target proteins and elucidating physiological mechanisms.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions involving elements of the protein translocatingsystem are employed in elucidating components of the system and theirfunction, modulating folding of proteins using chimeric genes employingan unnatural signal sequence to provide “conformers,” (proteins havingat least substantially the same amino acid sequence, but differentphysical topology or topography). By topology is intended the differentplacement of the protein, e.g. C-cytosolic as compared to N-cytosolic,and topography intends change in external conformation or shape,identifying conformers, providing in vitro and in vivo systems for thesepurposes, and identifying compositions that modulate proteinconformation during translocation. Topography as used herein refers toproteins of substantially the same or similar amino acid sequence butdifferent three dimensional shapes due to differences infolding/conformation. As used herein, polypeptides of substantially thesame amino acid sequence are those with conservative amino acidsubstitutions (i.e. a small or large side chain for a small or largeside chain, respectively; or an acidic, basic, polar or hydrophobic sidechain for an acidic, basic, polar or hydrophobic side chain,respectively), that do not alter the protein conformation or topology.Methods for identifying the multiple gene products that regulate signalsequence mediated selection of folding funnels during protein biogenesisalso are provided, using a reticulocyte lysate fractionation scheme. Themethodology for fractionation of translation extracts and fractionationof solubilized membranes with reconstitution of subfractions containingor missing particular trans-acting activities, offers several advantagesover existing systems. The process can be readily expanded to a largescale; the materials required for the fractionation are eitherinexpensive (common chemicals) or reuseable (centrifuge tubes, ionexchange resin); and the final products have a long shelf life whenstored appropriately.

This fractionated system offers several advantages over the currentlyavailable RRL, including: We have demonstrated that simply supplementingan S-100 fraction from Xenopus oocytes with RRL ribosomes results intranslation efficiencies more than an order of magnitude greater thanprevious oocyte translation systems (2). Such an oocyte system can beused to study oocyte specific events such as translational repression ofdevelopmentally regulated factors; and also, it appears that theribosomes contained in the rough microsome preparation from dog pancreasare highly active in restoring translation to the DEAE fraction. Thus,translocation events can be studied in an essentially homologous systemin which dog pancreas ribosomes are translating and translocatingproteins across dog pancreas membranes. Subtle and regulatedinteractions between ribosomes and translocon proteins may be morefaithfully reproduced in a homologous system versus a heterologoussystem.

This system can be readily adapted to other translation systems such asthe wheat germ translation system, or Xenopus oocyte translation system.In some cases, the fractionated system results in an enormous increasein the translation efficiency (see above). Furthermore, the ability tomix and match components from multiple such fractionated systems allowstissue specific events involved in the biogenesis of certain proteins tobe studied. This is potentially useful for the identification offactors, by complementation, involved in such tissue specific events.Examples of such tissue specific differences have been documented (Wolinand Walter, J. Cell Biol (1989) 109:2617-2622; Lopez et al., Science(1990) 248:226-229).

The proteins of interest are proteins, which have a signal sequence andare subject to processing in the endoplasmic reticulum. Numerous signalsequences have been identified from different proteins and appear to becapable of operating conjugated to a broad range of unnatural proteins.The signal sequences are usually N-terminal, but may be internal to theprotein or C-terminal. Signal sequences are selected from proteins thatare known to have a specific mechanism for translocation affecting theconformation of the product or may be synthetic, where thetranslocational effect is known or determined. It is now known that thesignal sequence affects the conformation of the protein that istranslated in conjunction with the translocon. Without being bound byany theory, the signal sequence directs whether the ribosome forms atight, loose or intermediate junction with the endoplasmic reticulum(ER) and the selection of the channel and accompanying processingproteins through which the translated protein is translocated andprocessed. For example, replacing a signal sequence of a protein with asignal sequence from preprolactin results in a tight junction., whilethe signal sequence from pre-β-lactamase provides a loose junction.Proteins which provide tight junctions include: growth hormone; andloose junctions include: immunological heavy chain and yeastalpha-factor; and intermediate junctions include ductin, calreticulin,PrP, angiotensinogen and MDR-1, where the division between the differentconformers may be attributed to a variety of mechanisms. (See, fordiscussion, Hegde and Lingappa, (1996) Cell 85, 217-228 for a discussionof the effect of the ribosome-membrane junction.) For a generaldiscussion of the mechanism of translocation, see, for example,Ellgaard, et al., (1999) Science 286, 1882-1888; Wickner, et al., (1999)Science 286, 1888-1893; and Ibba and Soll, (1999) Science 286,1893-1897.

Signal sequences may be rated by using a lysate competent forexpression, microsomes and proteinase K. The degree of proteolysisoccurring with different signal sequences and a common gene isindicative of the nature of the junction of the ribosome with the ER. Byusing signal sequences having different degrees of junction tightness,the conformation of the resulting protein can be modified. Thesedifferent conformers may be used in a number of ways. The conformers maybe used for the production of antibodies, either antisera or preferablymonoclonal antibodies. The antisera and antibodies are prepared inconventional ways using the different protein conformers to immunize ahost, usually a mouse in the case of monoclonal antibodies, with orwithout an adjuvant, followed by additional injections of the protein atbiweekly or longer intervals and monitoring the level of antisera. Formonoclonal antibodies, splenocytes may be isolated, immortalized andscreened. Those hybridomas which produce antisera which can distinguishbetween the conformers are expanded. A library is produced of antibodiesthat distinguish between the two conformers. The antibodies may then beused to isolate each of the conformers and assay for the differentconformers in hosts, using physiological samples appropriate to thenature of the protein.

Signal sequences may also be rated by analysis of crosslinking patternsgenerated when truncated transcripts encoding those signal sequences atthe 5′ end of the authentic coding region of interest are expressed bycell-free translation and subject to chemical crosslinking including butnot limited to lysine and cystiene specific cleavable and uncleavablecrosslinkers, with analysis of the crosslink patterns byimmunoprecipitation and polyacrylamide gel electrophoresis in sodiumdodecyl sulfate and subsequent autoradiography.

Different conformers may be screened by employing matrices of differentoligopeptides and/or oligonucleotides. See, for example, U.S. Pat. Nos.5,631,734; 5,856,102 and 5,919,523. These matrices are availablecommercially and can be prepared in relation to a particular bindingpattern. In this way, one can add a physiological sample and see whichof the conformers are present and estimate the amount of each. One mayalso use the matrix to isolate particular conformers by their bindingaffinity to the matrix. The matrix and antibodies may be used inconjunction to confirm the other assay, isolate the conformer, usedtogether in the same assay. In assays by themselves or in conjunctionwith other affinity binding assays, the antibodies may be labeled with adetectable label, e.g. fluorescer, luminescer, phosphorescer, enzyme,radioisotope, and the like. Numerous protocols are available fordefining specific epitopes, by which the conformers may bedistinguished. In some instances, it may be desirable to use two or moreantibodies, where the conformer may be defined by steric inhibition ofbinding, different affinity constants, or the like.

By virtue of the fact that the conformers can be distinguished byoligomers, particularly of oligopeptides, these oligopeptides may servefor identifying the different conformers. The oligopeptides can be usedin competitive assays for identifying other oligopeptides which competefor the site or other compounds, particularly small organic compounds,natural or synthetic, of less than 5 kDal, usually less than about 2.5kDal, which bind to the conformer. These compounds may then be used inturn to identify other compounds having greater affinity for the site.In this way drugs may be identified that are specific for one conformer,as compared to other conformers. The various binding entities may beused in assays, where the entity may be labeled to identify binding tothe conformer. The assays may be homogeneous or heterogeneous.

If desired, one may use random mutations of the wild-type and/orchimeric gene to express the resulting gene. The protein may be analyzedfor similarities and differences with the parent gene. Where the effectof the mutation is to change the conformer from one conformation toanother or to change an epitope, as determined by antibody binding. Orto generate differences in crosslinking pattern or conformation asscored by altered reactivity to chemical modifying agents undernon-denaturing condtions, including but not limited to N-sulfobiotin,trinitrobenzenesulfonic acid (TNBS), N-ethyl maleimide (NEM). Genes andthe proteins, which they express from human patients or animals, where amutation is naturally occurring or a result of mutating a gene, may becompared with the randomly mutated gene and its expression product in amammalian host. In this way, disease states resulting from mutationsthat lead to different conformers may be diagnosed and treatmentsdeveloped.

The conformers can be used for biomedical purposes in aiding in thediagnosis and treatment of patients. The conformers serve todifferentiate between individual hosts, who produce the conformers indifferent ratios, normally or under different environments. One of theenvironments is the presence of a compound, particularly a drug, and thebinding affinity and/or modulation of activity of a targetphysiologically active compound, usually a protein, such as a membranereceptor, channel, enzyme, transcription factor, housekeeping protein,cytoskeletal protein, membrane protein, and the like. The conformers maybe mobile or immobile proteins, being bound to membranes or free insolution. A compound would be mixed with the different conformers, inthe same or different vessel, and the binding of the compound to theconformer determined. The determination may be made in a variety ofways, either competitive or non-competitive. The amount of compoundremaining in the solution may be determined, where a reduction in amountwould be indicative of the binding of the compound to the conformer.Alternatively, a labeled compound which binds to the conformer may beused, where interest in binding is as to a particular region of theprotein, such as in the case of enzymes and receptors, as well as otherproteins where particular sites are associated with biological activity,e.g. G-proteins, transcription factors, DNA binding proteins, etc.

The conformers are also useful in determining differences in activity inrelation to their physiological activity. The binding affinity for theirbinding partners can be determined in assays, either homogeneous ornon-homogeneous, which allows for evaluation of the level of activity ofa mammalian host in relation to the proportion of the two conformers.Again, numerous protocols are available for detecting binding between aligand and a receptor or two or more proteins involved in complexformation. The two proteins may be labeled with a fluorescer and anenergy receptor, so that binding of the two proteins together wouldreduce the level of emission at the wavelength of the fluorescer andincrease the emission at the level of the energy acceptor.Alternatively, one may bind one of the proteins to a surface and thelevel of binding of the other protein to the bound protein determined byhaving the second protein labeled. Alternatively, one may bind each ofthe proteins to different particles, where one particle produces acompound, which activates the other particle, such as LOCI. Where thelabeled entity is small, such as an oligomer or small organic molecule,a fluorescer may be used as the label and fluorescence polarizationemployed for detection. Illustrative of assays are the assays describedin U.S. Pat. Nos. 5,989,921; 4,806,488; 4,318,707; 4,255,329; 4,233,402;and 4,199,559.

In determining the presence of conformers when screening physiologicalsamples, the samples may be blood, cells, csf, saliva, urine, hair, etc.The sample may be subject to pretreatment, such as adding citrate toblood, coagulating and separating erythrocytes, dilution, extraction,etc. Thus, the conformers may be identified as being associated withparticular indications, which may relate to diseases, response to drugs,physical performance, cellular degeneration, apoptosis, etc.

Illustrative of the power of the subject invention is the investigationof prions. It is found that by varying the signal sequence one canobtain the natural conformation ^(sec)PrP, and two other conformations,^(Ntm)Prp and the neurodegenerative ^(Ctm)Prp, in varying amounts. Thisaids in the determination of the mechanism of the change in theproportion of formation of the neurodegenerative form as compared to thewild-type form. In an analogous manner, one may vary the signalsequences for other proteins having signal sequences, where the proteinis suspected of being associated with a diseased state or loweredperformance. One could then determine whether the protein can beproduced in varying conformations. If different conformers are found, asdescribed above the presence of the different conformers is establishedin healthy normal patients as compared to patients who have the diseaseor reduced performance. Drugs would then be screened as described aboveagainst the undesired conformer.

In addition, by using different signal sequences one can investigate thebasis for the change in proportion between the desired conformer and theundesired conformer. In conjunction with, in addition to or in place of,one can use in vitro lysates for investigating the role of ER associatedproteins with the formation of the conformers. Microsomes are preparedlacking all but the essential proteins for translocation. The individualproteins may then be replaced individually or in combination todetermine the effect of the presence of the protein(s) on the formationof the conformers. Once the protein(s) involved in the formation of theconformers is determined, one can screen healthy and abnormal patientsfor the presence of the protein(s) involved in the formation ofconformers and determine the presence of mutations or differentconformers. In this manner, one not only elucidates the mechanism bywhich proteins fold in the translocon, but also establish new targetsfor therapies.

For the cell-free protein translation mixture, various systems may beemployed (Erickson and Blobel, Methods Enzymol. 96:38-50 (1983);Merrick, Methods Enzymol 101:606-615 (1983)). Illustrative are wheatgerm extract and rabbit reticulocyte extract, available from commercialsuppliers, e.g. Promega (Madison, Wis.), as well as high-speedsupernatants thereof. Other references include U.S. Pat. Nos. 5,998,163;5,998,136 and 5,989,833. Such systems include, in addition to thecell-free extract containing translation machinery (tRNA, ribosomes,etc.), an energy source (ATP, GTP), and a full complement of aminoacids. Methods known in the art are used to maintain energy levelssufficient to maintain protein synthesis, for example, by addingadditional nucleotide energy sources during the reaction or by adding analternative energy source, e.g., creatine phosphate/creatinephosphokinase. The ATP and GTP present in the standard translationmixture will generally be at a concentration in the range of about 0.1to 10 mM, more usually 0.5 to 2 mM. Generally, the amount of thenucleotides will be sufficient to provide at least about 5 picomolar ofproduct, preferably at least about 10 picomolar of product.

Lymphocytes also form PrP and may be used as a screen for the effect ofagents, e.g. compounds as candidate drugs or as a screen for response ofan individual to changes in the environment or physical insults. Thelymphocytes would be subjected to a change in the environment, whichcould be a chemical change, e.g. addition of a compound, a physicalchange, e.g. change in pH, etc., where the sensitivity of the particularcells to the change would be determined as a measure of the propensityof the person to respond to the environmental change by changing thenature of the PrP. By determining the response to changes in theenvironment, which could include pollutants, pesticides, etc., one candetermine whether the compounds are a general threat or only affectidiosyncratic people. The cells may also be used to determine whether apatient who is symptomatic for a neurodegenerative disease has a Ctm-PrPrelated disease by determining the presence and/or level of Ctm-PrP inthe lymphocytes as compared to normal individuals. Numerous assays maybe employed as described herein to determine the presence and level ofCtm-PrP. By detecting a propensity for neurodegenerative disease, thepatient could be directed away from activities or exposure to compoundsthat might increase the probability of the neurodegenerative disease. Inaddition, the effect of compounds on Ctm-PrP may be used as a substitutein relating the activity to other diseases that respond in an analogousway. The cells may also be used for individual patients to evaluate theresponse of the individual to various drugs, determining the effect ofthe drug on the production of Ctm-PrP

As described above, many proteins have-different translocationaloutcomes. Proteins known to have alternatives in the translocationalpathway, such as existing in two different conformers, the presence ofdifferent methionines that may be selected as the f-Met, resulting in adifferent signal sequence, having hydrophobic regions of from about 15to 20 amino acids in the chain, which can be putative transmembranesequences, but are available as secreted proteins or are transmembranethat are secreted can be used as markers for the effect of compounds ontranslocational outcome. Illustrative proteins include ductin,calreticulin, MDR-1, and PrP.

Numerous proteins are associated with the ER in the process oftranslocation, frequently being part of the ER and associated with thechannel through which the nascent protein is transported to the ERlumen. These proteins include the ribosome, proteins of the signalrecognition particle (SRP), the heterotrimeric Sec61complex with α, β,and γ-subunits, translocating-chain-associated membrane protein (TRAM),signal peptidase (a complex of five proteins), oligosaccharyltransferase (a 3-protein complex), ER lumenal proteins, including BiP,GRP94, calnexin (CNX), ERGIC-53, protein disulfide isomerase (PDI),Erp57. Erp72, chaperones, such as Hsp 90, hsp 47, Hsp 60 or GroELfamily, Hsp 70 or DnaK family, Hsp 100 or Clp family, and heat shockcognate 70, tapasin, microsomal triglyceride transfer protein,protective protein/cathespin A, β-catenin, egasyn, co-chaperones, suchas proteins from the DnaJ family, enzymes, such as uridine5′-diphosphate (UDP)—glucose:glycoprotein glucosyltransferase (GT),glucosidase II, prolyl-hydroxylase and carboxylesterase.

The ancillary proteins associated with the ER and translocation of thetranslocation product may be removed from the lysate by methodsdescribed in the literature. (Gorlich, et al., (1992) Nature 357, 47-52;Gorlich and Rapoport, (1993) Cell 75, 615-630; and Hanein, et al.,(1996) Cell 87, 721-732.

By varying the signal sequence in proteins of interest, one can preparegenes for expression in mammalian hosts. Methods of replacing a DNAsequence to provide a chimeric gene are legion today. See, for example,Sambrook, et al. (1989), A Laboratory Manual, Second edition, ColdSpring Harbor Press. Briefly, the DNA for the gene is isolated knowingthe amino acid sequence and using degenerate probes. The DNA sequences,which bind to the probes, are isolated and sequenced to see the presenceof a sequence coding for the protein of interest. If one wishes to avoidthe presence of introns, the mRNA may be isolated, reverse transcribedand amplified using PCR. The signal sequence in either situation may bereplaced with a different signal sequence by amplification using onepair of primers, with one primer having a signal sequence at its5′-terminus joined to a sequence complementary to the sequence of thegene contiguous with the native signal sequence the other primercomplementary to the first primer. A second set of primers will providethe other terminus of the DNA sequence. Depending on the size of thegene, one may select a convenient restriction site for linking themodified 5′-terminus DNA with the remainder of the coding sequence.Various techniques are available and each gene will have an obviousselection of protocols to enhance the convenience of the particularsynthesis.

Once the gene is produced it may be introduced into one of numerouscommercially available vectors and cloned and/or an expression vectormay be employed having a transcriptional regulatory region 5′ of thesense strand to provide for expression. By using mammalian cells, theconformers from the different constructs can be produced and isolatedand assayed as described above. In this way, significant amounts of thedifferent conformers may be isolated.

In an initial stage, one may wish to concentrate the conformers usingdifferent separation techniques, such as HPLC, capillaryelectrophoresis, affinity chromatography, etc. The initial separationmay solely serve to enhance the concentration of the conformers in aparticular fraction or may provide for separation of the conformers. Inthe former case, one would need further separation of the conformers,using techniques, which have been described above. Various separationmedia may be employed, involving ion exchange, sieving media, affinitymedia, etc., using different eluents to establish procedures forseparation and isolation of the different conformers. Various procedureshave been developed and each set of conformers will use protocols thatoptimize the separation with minimum denaturation. These protocols maybe readily identified by those of skill in the art of proteinpurification. The purified conformers may then be used for assays, forx-ray crystallography, to identify differences in structure, the aminoacids associated with the different epitopes, the interactions withproteins with which the wild-type protein is associated, as well asother proteins to which the conformer may bind, and the like.

Once having the gene for the chimeric protein, the gene can be used ingene therapy, mutating laboratory animals, random mutation to determinethe effect of mutations on folding, in expression constructs and singlecell or organism host for large scale production of the protein, and thelike. Laboratory animals include rodents, lagomorpha, birds, canine,feline, porcine, etc.

In some instances, one may create viral constructs, where the chimericgene is introduced into a viral carrier for introduction into cells,either randomly or where the virus has a tropism, into cells for whichthe virus is tropic. Where the gene has a beneficial effect, even in thepresence of the other conformer, the presence of the construct may serveto alleviate the symptom resulting from the native conformer.Alternatively, the introduction of the non-native conformer will allowan evaluation of the effect of the non-native conformer on thephysiology of the cell, tissue, organ and host.

Of particular interest is the introduction, preferably replacement ofthe wild-type gene with the chimeric construct comprising the wild-typestructure and a different signal sequence that is known to result in adifferent conformer. By using homologous recombination with germ cellsor a nucleus that is subsequently transferred to a germ cell, one canprovide for the expression of the desired conformer(s). Whereheterozygosity is involved, the progeny having the chimeric gene may bemated to obtain homozygous progeny. In this way, one can investigate therole of the conformer(s), whether the location(s) to which the proteinis transported changes, the effect of such variation in location, theeffect of the different epitopes or topography on the function of theprotein, and the like. In addition, the chimeric mammal may be used toscreen compounds for their biological effect on the conformer, so thatdrugs which are directed to a particular indication, which involves theconformer, either directly or as a result of the conformer being in thepathway, can be evaluated. The animal models may be used todifferentiate the effects on the wild-type conformer and otherconformers that can be formed.

In situations where the non-native conformer is a dominant negativeallele, the loss of activity can be monitored as to the effect on thephysiology of the host. The effect on the host can be compared todisease states, where similar indications are observed. Treatments canthen be evaluated by employing drugs having known function associatedwith the indication, compounds known to bind to the wild-type allele orother treatment to evaluate the effectiveness of the treatment. Cellshaving the unnatural conformer may be screened for the effect of theunnatural conformer on the expression of other proteins. By convertingthe mRNA to cDNA of such cells, one can by using various subtractiontechniques known in the literature, between the cells without theunnatural gene and the cells with the unnatural gene, determine theeffect of the unnatural gene on the expression of other proteins.Alternatively, one may express the various proteins and compareelectrophoretic, mass spectrophotometric or HPLC patterns to determineif the different conformers affect the expression pattern of the host.

Following the procedures described in the above description and in theexperimental section, or other functionally equivalent procedures, thesubject invention allows for investigation of diseases associated withthe prion protein and analogous diseases, particularly diseasesassociated with topological modifications as in Alzheimer's disease. Asshown, by preparing expression constructs for expression of the nativePrP, the Ntm-PrP and the Ctm-Prp, one can modify cells and animals toproduce the different forms of the protein. In this way, one can studyin culture and in vivo the effect of the addition of various candidateagents and determine the change in the formation of the differentconformers in the presence of the agents in culture and in vivo. Thesubject proteins allow for the controlled presence of the differentconformers and a study of the etiology of the disease, the otherentities involved in the etiology of the disease, by doing expressionanalysis of the effect of the different conformers on expression in thesame and different cells and on the physiology of the host. Mouse modelsmay be developed with the construct in the presence or absence of thenative PrP gene, since the host gene may be knocked-out and replacedwith the chimeric gene. Alternatively, mutations may be introduced intothe PrP gene that result in a modification of folding and a modificationin the dependence on or interaction with proteins associated withtranslocation. Thus, the signal sequences, either native or chimeric tothe gene, may be mutated and the effect on translocation-relatedproteins determined.

Uses of the Invention

The invention finds use at the molecular, cellular and organismal levels(i.e. in elucidating, diagnosing or treating a disease). Following areprovided specific applications.

1. Using the described invention, it would be possible to manipulateantibodies to increase their functions and uses. By signal sequencereplacements, alone and in conjunction with fractionated andreconstituted or variant membrane and cytosols in cell-free translationsystems, the pathway of folding of an immunoglobulin could be altered ineither subtle or profound ways, without actually changing the amino acidsequence of the final product itself. Thus, it would be possible toachieve variant immunoglobulins in which the binding constant wereincreased or decreased incrementally, and immunoglobulins with changesin their effector functions (e.g. the binding specificity of their Fcregion). Current approaches to achieve these ends (e.g. by site-directedor random mutagenesis of the authentic immunoglobulin chains) have thedisadvantage of changing not only the folding pathway but also theprimary structure of the molecule itself. Our approach would, in effect,dissociate this two types of changes, allowing one without the other,with diagnostic and/or therapeutic utility. The signal sequencevariations could also be applied to transfected mammalian cells,allowing medium to be harvested and screened for conformational andfunctional differences.

2. Most hormones, cytokines, and growth factors have been implicated ina wide range of functional behaviors, and in some cases, activation ofmultiple signaling pathways. For example, leptin, first discovered as ahormone that signals satiety, has been found to serve as an independentcentral regulator of bone density, and to be implicated in wound healingand control of blood pressure. Some of these functions clearly occur inthe absence of the others, and most obese individuals are leptinresistant, without having disorders of bone density, suggesting that theleptin they make is not able to serve the satiety promoting function,but is able to function in maintenance of bone density. The conventionalassumption has been that this sort of heterogeneous action is achievedthrough diversity in receptors or receptor function. However, diversityof ligand conformation, as demonstrated in the examples from PrP andprolactin shown here, could account for some or even most of theheterogeneity of outcomes, in the case of leptin.

By allowing individual conformers to be identified, and pharmaceuticalsto be developed to increase or decrease the expression of individualconformers, or to block action of one conformer or another, it should bepossible to affect gene expression in ways that promote some functionsof hormones over others. It should also be possible to stratify patientsinto subsets based on the mix of conformers that is manifest with theirparticular genotype and phenotype. This would in turn, allow differenttreatments to be administered to different individuals taking intoaccount the mix of conformers they are expressing at that particularpoint in time, using different pharmaceutical agents designed or shownto be most efficacious for a particular conformer mix or otherconformer-related subset.

Thus, in the case of leptin, swapping of signal sequence, and synthesisin various combinations of translation systems containing fractionatedcytosol and fractionated and reconstituted or variant membranes, andsubsequent screening programs should make it possible to generate formsof leptin that would promote bone density without affects on satiety orwound healing, or vice versa. Likewise, forms of leptin that would bemore active at appetite suppression and thereby overcome leptinresistance, the most commonly observed phenotype in obese humans, couldbe generated. Conversely, small molecule pharmaceuticals could bedeveloped, by screening for their effect on individual subsets of leptinconformers, or for their affect in altering the mix of leptin conformerssecreted. Thus it would be possible to develop drugs that haveconformer-specific or selective effects, thereby blocking or enhancingthe effects of conformers, or blocking or enhancing the body's abilityto make a particular mix of conformers. Half-life, association withother molecules, secretion kinetics are parameters besides affinity forparticular receptors that could be altered by signal sequencemanipulations without altering in any way the sequence of the mature,authentic protein. This approach would apply also to secretory andintegral membrane enzymes, where substrate turnover, rather thanreceptor affinity is the relevant functional parameter.

3. This approach can be combined with transgenic technology to introducevariant genes differing in their signal sequences, into wild-type andknock-out mice (lacking an endogenous gene). In this manner it will bepossible to screen for functions of complex secretory and integralmembrane proteins that currently are not known. Thus it is known that95% of the wild-type Cystic Fibrosis Transmembrane Regulator (CFTR) isdegraded immediately upon synthesis. The conventional assumption is thatthis represents “errors” in biogenesis. An alternative interpretation,for which the present invention has utility, is that these 95% representthe sum total of many alternative conformations that are not needed bythe cell at one particular time, but may be rescued from degradation atparticular times in development. Since the signal sequences of CFTR arenot cleaved, the goal of altering folding without introducing mutationsmust proceed exclusively by regulation in trans rathe than in cis. Thus,expression of CFTR in translation systems complemented with fractionatedand reconstituted or variant membranes and fractionated andreconstituted cytosol, as claimed, would enhance one versus anotherconformation, which would in turn be scored and catalogued by monoclonalantibody reactivity, chemical modifications, crosslinking and otherproperties, and then introduced into transgenic animals and screened fornovel phenotypes and disorders. Similar approaches can be taken for eachof the channel forming and receptor forming membrane proteins, includingboth single and multispanning with respect to the ER and/or otherinternal and/or plasma membranes.

4. The most difficult to currently treat diseases in humans aredisorders of signaling as typically manifest by hormone, growth factor,cytokine and receptor resistance (also known as desensitization or downregulation). Many different mechanism of resistance have beenidentified, whether most cases of most diseases are due to themechanisms identified or due to other as yet unknown mechanisms remainsto be determined. Based on our studies with PrP as summarized in ourpublished and unpublished work, we believe that conformationaldysregulation (the wrong mix of conformers being made or being allowedto exit from the ER) is a central mechanism of disease pathogenesis.This can be demonstrated by raising monoclonal antibodies to secretoryproteins and demonstrating that individual subsets of the secretoryproteins are present (or absent) in a disproportionate manner in adisease state or in subsets of patients with a disease state. The majorgenes for the major diseases afflicting humans include a large number ofsecretory and membrane proteins, which could be screened for theinvolvement of conformational dysregulation in their pathophysiology.Diabetes, hypertension, hyperlipidemia, obesity, osteoporosis,degenerative joint disease, cancer, Alzheimer's disease, and psychiatricdisorders are just a few examples already implicated in this fashion.

Application of the Invention to the Research and Treatment of Disease

As listed above, a number of important medical conditions involve genesfor which conformational regulation is likely to be an importantdimension of physiological and pathophysiological function. Here it isdescribed how the invention is applied to better understanding specificdiseases.

First, key disease-related genes that are secretory and integralmembrane proteins would be analysed in cell-free systems and intransfected mammalian cells, to characterize the class in which theirsignal sequences fall. The conformational heterogeneity of the nativeproteins will be catalogued both in vitro and in vivo, including insamples from mice and humans (e.g. from blood), if available, usingmonoclonal antibodies, chemical modifications and co-association withother proteins inside the cell and in the medium, as the means ofscoring conformation.

Second, attempts will be made to skew the conformational mixsynthesized, allowing minor and transient conformers to be magnified andstabilized and therefore more readily detected and characterized, sothat they can be distinguished from the normally dominant conformers.This can be done in two general ways, namely by swapping signalsequences that are cleaved or by expressing the proteins in fractionatedand reconstituted systems that modify the native conformer mix throughactions (or their absence) in trans.

Third, once the mix of conformers that define the key disease-relatedproteins has been characterized, samples from patients representing thediversity of the natural history and phenotypic classes of the diseasein question, will be screened and categorized with respect to theheterogeneity of conformers of these proteins observed. From thisanalysis it will be possible to identify i) the conformers implicated indisease; ii) changes in conformer mix that precede actual development ofdisease, iii) changes in conformer mix that stratify individuals withrespect to disease progression, complications and other aspects ofnatural history, including increased or decreased risk of drug efficacy,side effects and other reactions. Note that these are all goals ofconventional proteomics programs which will be missed by those programsbecause they are not aware of the evidence, submitted in support of thesubject invention, for conformational heterogeneity of proteins. Hencethey are not looking for alternate conformers of the protein inquestion. Furthermore, without the subject invention, there arecurrently no means of systematically identifying, magnifying, andcharacterizing these changes in conformers, without which it isimpossible to determine their distribution or significance inpopulations at risk of, or afflicted with, particular diseases.

Fourth, with a variety of valuable information on disease association ofconformers in hand from 1-3 above, it is possible to develop assays asoutlined previously to screen for agents that modify the conformer mixin a way that minimizes undesired conformers and maximizes those thatare protective or not associated with disease progression, drugtoxicity, etc. Likewise, agents that block undesired conformersselectively, or that enhance the action of desired conformers can besought through high throughput screens of large compound libraries, aswell as through conformer-specific rational drug design.

Specific Applications to some Major Diseases are Outlined Below.

Diabetes mellitus: The key genes for insulin, the insulin receptor andthe many members of the family of glucose transporters all have cleavedor uncleaved signal sequences that make them amenable to this analysis.Furthermore, a key pathophysiological process impacting on patient careis the syndrome termed insulin resistance. Usually this is attributed todisorders in receptor function, but in most cases, the basis for thedisorder is unknown, and the possibility that the disorder could derivefrom the ligand rather than the receptor, cannot be ruled out. In thiscase, the ligand is insulin, and the aberrant conformation that impairsinsulin-mediated signaling could be a property of either insulin or theinsulin receptor or individual members of the family of glucosetransporters, which could be identified in the manner described.Finally, the huge heterogeneity of individual phenotype at any giventime, and in natural history over time, which has remained elusivedespite massive research efforts directed at conventional genomic andproteomic analysis, is strongly suggestive of disordered conformatics,amenable to the subject invention.

Hypertension: A number of key genes in control of blood pressure encodesecretory and integral membrane proteins including the epithelial sodiumchannel (EnaC), angiotensinogen, the angiotensin receptors, renin, theenzymes of steroid biosynthesis involved in synthesis of aldosterone andother steriods, and the alpha and beta adrenergic receptors, to name afew. The pathophysiology of hypertension remains largely mysterious,hence the possibility that conformer dysregulation plays a role in itsetiology and pathogenesis cannot be excluded in any way. Clinicalepidemiological and observational studies clearly indicate that patientsare heterogeneous not only with respect to the nature of theirhypertension-inducing state (e.g. salt sensitive vs salt insensitive;increased sympathetic activity, etc.) but also with respect to theirsensitivity to individual classes of drugs, side effects of those drugs,and ability to tolerate the drugs.

Obesity: The key genes involved in the control of obesity, includingleptin and its receptors, and the various obesity related genesidentified to date including the melanocortin and mahogany receptors,the agouti protein, etc. all contain cleaved or uncleaved signalsequences. Furthermore, most obese individuals are leptin resistant,consistent with the hypothesis that conformer differences of leptinmediate its diverse physiological functions (e.g. in satiety,maintenance of bone density, promotion of wound healing, and regulationnof blood pressure).

Cancer: Conceptually, cancer is a disorder of signaling pathwaysinvolved in cell growth and proliferation and the physiological controlsover these processes, including a host of diverse apoptotic triggers andantiapoptotic factors. Many of these are secretory or membrane proteinswith cleaved or uncleaved signal sequences. In many cases, cancer can beaffected, both positively and negatively, by a host of secreted growthfactors and cytokines, which also generally have signal sequences. Ahost of data supports the notion that many of these factors aremultifunctional, or more precisely, are associated with both promotingand inhibiting particular signalling pathways. A present conundrum inthe field is understanding how one factor can bring about one action atone point in time, and yet bring about a very different action atanother time. A switch from one to another conformer resulting inaltered receptor interaction, signal transduction, half life,associations etc., could be central to one or more pathways ofcarcinogenesis and phenotypic variation in the development, immunesurveillance, presentation and progression of cancer, or in response toits treatment.

Osteoporosis: The hormones involved in the regulation of bone density,including leptin discussed earlier, and others such as parathyroidhormone, osteoprotegenin, and their receptors, all have signalsequences, some cleaved, some uncleaved, rendering them amenable toanalysis by the subject invention. The physiology of bone density iscomplex, poorly understood and subject to a variety of confoundingparadoxes—the same agent that promotes bone density in somecircumstances can result in bone loss under others. Conformationalheterogeneity in response to signaling changes is a potential basis forthese observations, with broad potential for diagnostics andtherapeutics.

Neurodegeneration: Including prion and alzheimer's diseases, spinal cordinjury, and stroke, are areas of perhaps the greatest potential fordiagnosis and therapy involving conformatics. Prion protein, the geneproduct responsible for prion diseases was the first protein for whichconformational regulation was demonstrated. In this case, conformationalregulation is manifest as topological regulation—the three detectableconformers differ in transmembrane topology, making them relatively easyto identify and distinguish. Amyloid Precursor Protein, the gene productwhose aberrant metabolite has been implicated in Alzheimer's disease, isan integral membrane protein which has features in common with the prionprotein, including a cleavable signal sequence. Netrins, semiphorins andother genes implicated in axonal guidance (and therefore central torecovery from spinal cord injury), have paradoxical activitiesconsistent with the hypothesis of multiple conformers.

Coronary artery disease: Apolipoprotein B is a complex, multifunctionalsecretory protein involved in low density lipoprotein metabolism and aprime candidate for conformational heterogeneity. Essentially everyintegral membrane channel and receptor protein in the body haveuncleaved signal sequences amenable to fractionation as describedelsewhere.

Chronic obstructive pulmonary disease: The Cystic Fibrosis TransmembraneRegulator (CFTR) is a multispanning integral membrane protein discussedpreviously, whose biogenesis suggests a more complex fate that isgenerally accepted. One reason for the difficulty in acceptance of thehypothesis that degraded chains of CFTR represent alternateconformations not needed at that point by the cell, rather than truemisfolded chains, is that our evidence in favor of conformatics remainslargely unpublished. While those alternative functions remain unknown atthe present time, it has been speculated that, the known function ofCFTR as a chloride channel affects the salt environment needed foranti-microbial peptides such as the defensins, needed for innateimmunity against pathogenic microbes. Thus, conformational dysregulationin which chloride channel function is lost may confer increasedsusceptibility to infection in the lung and other tissues, as isobserved in a wide range of pulmonary and other disorders. By usingfractionated cell-free translation and translocation systems, asdescribed in this invention, in combination with available scoringsystems including monoclonal antibodies, chemical modificationfingerprints, crosslinking and association analysis including sucrosegradient studies, we can catalogue the alternative conformations ofCFTR, MDR genes, and other relevant transporters and determine whetherthese alternatives are utilized to a greater or lesser extent inparticular subsets of patients with infectious and other disorders.

Psychiatric disorders: As with neurodegeneration, functional disordersof the brain are likely to involve disturbances in signaling and/orsignal transduction. Since receptors on the cell surface, including thefamily of G protein-coupled receptors, are generally responsible forcell to cell signaling and generally have signal sequences, cleaved orotherwise, these classes of proteins are highly likely to be candidatesfor conformational regulation.

Taken together, the examples cited above demonstrate the broad nature ofthis invention and its applicability to a wide range of disorders inwhich a signal sequence containing protein or receptor is involved.

Relevant manuscripts accompany this application, were submitted as partof priority applications 60/171,012 and 60/172,350, and are included byincorporation by reference, as if they were specifically set forthherein. These manuscripts are identified by their title and firstauthor: Rutkowski, et al., “A New Role for the Signal Sequence inTranslocational Regulation”; Hegde, et al., “Transmissible and GeneticPrion Diseases Share a Common Pathway of Neurodegeneration” (publishedin Nature (1999) 402:822-826); and Lingappa, et al., “ConformationalControl Through Translocational Regulation: A New View of Secretory andMembrane Protein Folding.”

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Materials

Rabbit reticulocyte lysate (RRL) and dog pancreatic rough microsomeswere prepared and used as described (Hegde and Lingappa, Cell (1996)85:217-228, and references therein). Mouse brain microsomes wereprepared in the same manner as canine microsomes. PK was also preparedas described (Hegde et al, Cell (1998) 92:621-631. Anti-prolactinantibody was purchased from USB (Cleveland, Ohio) and 3F4 monoclonalanti-PrP antibody was a gift from the Prusiner laboratory. Anti-PDI waspurchased from StressGen (Victoria, BC). Disuccinimidyl suberate (DSS)was from Pierce (Rockford, Ill.). Saponin was from Calbiochem (La Jolla,Calif.) and was dissolved as a 20% w/v stock in water, adjusted to 10 mMHepes, pH 7.2, and the contaminants removed by passage over a 1.5 mlcolumn (per 10 mls saponin) of SP-sepharose fast flow (AmershamPharmacia; Piscataway, N.J.) and a 2 ml column of Q-sepharose fast flow.A clone encoding yeast prepro-alpha factor was provided by Tom Rapoport.

Fractionation of Reticulocyte Lysate.

The process described herein is for the preparation of a modified invitro translation system derived from the presently available rabbitreticulocyte lysate (RRL) translation system. The RRL translationsystem, originally developed by Jackson and Hunt (Methods Enzymol (1983)96:50-74), offers a mammalian based extract competent for translation ofmessanger RNA (either synthetic or native). Furthermore, whensupplemented with microsomal membranes, the biogenesis of secretory andmembrane proteins can be reconstituted. By fractionating the RRL intodefined components, we have extended the flexibility and potential usesof the in vitro translation system. These advances have severaladvantages for addressing a variety of biological phenomena (see below).Briefly, RRL is separated into native ribosomes, and a soluble proteinfraction (S-100). The S-100 is further fractionated by anion exchangechromatography on DEAE sepharose. The flow-thru (containing all of theglobin) is discarded, and the column step-eluted with 300 mM KCl. Theeluate (which contains the relevant translation factors) is concentratedby ammonium sulfate precipitation followed by dialysis. This DEAEfraction, along with the ribosomes can reconstitute translation withessentially equal efficiency as the starting RRL.

The following is the detailed protocol for preparation of the fractionsfrom 1 ml of RRL. It can easily be scaled up as necessary. Column bufferis 20 mM Tris-Acetate, pH 7.5, 20 mM KCl, 0.1 mM EDTA, 1 mM DTT (addedfresh), 10% v/v Glycerol. Elution Buffer is same as Column Buffer, butwith 300 mM KCl. Dialysis Buffer is 20 mM Hepes-KOH, pH 7.5, 100 mMKOAc, 0.5 mM MgOAc, 0.1 mM EDTA, 1 mM DTT (added fresh), 10% v/vGlycerol. Fractionation protocol: i) RRL is prepared according topreviously published protocols (Jackson and Hunt, supra), except that itis not desalted. Briefly, blood cells from an anaemic rabbit are washedseveral times, and the cytosol released by hypotonic lysis. The unlysedcells and ghosts (cells which have lysed and released their cytosol) areremoved by centrifugation. The supernatant is the RRL used in thisfractionation. ii) RRL is adjusted to 1 mM CaCl₂, digested with 150 U/mlmicrococcol nuclease for 10 minutes at 25° C., and the reactionterminated by the addition of EGTA to 2 mM. The RRL is chilled to 0° C.on ice, and all subsequent procedures done at 4° (in the cold room) oron ice. iii) The nucleased RRL is centrifuged for 20 minutes at 100,000RPM in thick-walled polycarbonate tubes (1 ml capacity) in a TL-100.2rotor at 4° C. The supernatant (S-100) is removed to a pre-chilled 2 mleppendorf tube on ice. The pellet is rinsed once in Dialysis Buffer, andresuspended in 100 μl of dialysis buffer. The ribosomes are then frozenin aliquots and stored at −80° C. They are stable for at least one yearwithout any loss of activity. Avoid excessive freeze-thaws, although itappears stable to at least 2-3 uses without any problems. iv) The S-100is diluted with an equal volume (1 ml) of column buffer and applied to a3 ml DEAE sepharose column. The column is washed with additional columnbuffer until the last traces of the bright red globin have flowedthrough. The column is then step eluted with 10 mls of Elution Buffer,and eluate collected. To the 10 mls of eluate, 5.6 grams of solidammonium sulfate is added slowly with constant stirring (bringing it to˜80% saturation). Stir at 4° C. (or on ice) for an additional 20-30minutes after the last of the ammonium sulfate is added. Sediment theprecipitate by centrifugation (10,000×g for 15 minutes), remove anddiscard the supernatant, and dissolve the pellet in at most 1 ml ofdialysis buffer, preferably 0.5 ml (it goes into solution readily withonly gentle mixing). Dialyze against 500 ml dialysis buffer to removethe residual ammonium sulfate (with one change of buffer if desired) at4° C. for 8-12 hrs. The sample is recovered from the dialysis tubing,frozen in aliquots, and stored at −80° C. It is stable for over 1 yearwith no noticeable loss of activity. Avoid excessive freeze-thaws,although it appears stable to at least 2-3 uses without any problems.

Example 1 Prion Protein

A dramatic example of a substrate with complex and highly regulatedtranslocation is the prion protein (PrP), a 35 kD brain glycoproteininvolved in the pathogenesis of several neurodegenerative disorders(Prusiner (1997) Science 278, 241-251). PrP is simultaneouslysynthesized in three alternate topological forms at the ER (Hegde etal., (1998) Science 279, 827-834). One of these forms, termed ^(sec)PrP,is fully translocated across the ER membrane, and is the predominantform observed in vivo. By contrast, the other two forms of PrP are madeas singly-spanning membrane proteins in opposite orientations witheither the N- or C-terminus in the ER lumen (termed ^(Ntm)PrP and^(Ctm)PrP, respectively). PrP biogenesis appears to involve multiplesteps leading to at least three distinct and assayable endpoints, ittherefore was used as a sensitive probe for any potentialsubstrate-specific effects of N-terminal signal sequences. In thefollowing experiments, the native signal sequence of PrP was replacedwith the well-studied signal sequences of the secretory proteinspreprolactin and pre-beta-lactamase (FIG. 1A). The chimeric proteins(Prl-PrP and βL-PrP, respectively) were compared to native PrP for theirability to be targeted, translocated, and synthesized in each of thetopological forms characteristic of PrP (FIG. 1B).

Plasmid Constructions

Standard techniques were used in the creation of all plasmid constructs(Sambrook et al 1989). All constructs were made in the pSP64 vector(Promega, Madison, Wis.) containing the 5′ UTR of Xenopus globininserted at the HindIII site. Prl-PrP was constructed by PCRamplification of the region encoding amino acids 1-30 of preprolactinand amino acids 23-28 of PrP. The PCR product was ligated into aBg12-PflmI digested vector containing wild-type PrP (PrP SV12), so thatthe resulting clone contained a precise fusion of the preprolactinsignal sequence to the mature region of PrP. All other PrP, AV3, andG123P signal sequence replacements were constructed similarly, exceptthat for Prl-PrP_((AV3)), the site of fusion was engineered three aminoacids downstream of the βL signal cleavage site. This clone wasidentical in final topology to Prl-PrP_((AV3)) constructed as a perfectfusion. The μL signal sequence was found to contain an Asp at position 2rather than Ser. This replacement had no significant effect on βL-PrPtopology. PrP_((R2,3)); PrP_((R4,5)); PrP_((D2,3)); and PrP_((D4,5))were created by first introducing a silent NheI site at codon 8 of PrPSV12, and then ligating annealed oligos encoding these mutations asBg12-NheI fragments. To fuse these signals sequences to the prolactinmature region, the latter (beginning at amino acid 34) was PCR-amplifiedand digested with NcoI. This fragment was ligated into the wild-typepreprolactin vector (pSP BPI) digested at NcoI, effectively deleting thefirst 33 amino acids of prolactin. An XbaI site, encoding Thr-Arg, wasengineered immediately upstream of amino acid 34, and PrP, preprolactin,pre-beta-lactamase, and prepro-alpha factor signal sequences wereamplified and inserted as HindIll-SpeI fragments. Prl-βL was constructedusing an identical scheme. All clones were verified by dideoxysequencing.

Cell-Free Translation and Proteolysis

In vitro transcription with SP6 RNA polymerase, translation with RRL,and translocation into canine rough microsomes have been described(Hegde et al, (1998) Cell 92, 621-631 and references therein).Translations were carried out at 32° (or 26° in FIG. 4C) for 20-45minutes. Where indicated membranes were isolated by sedimentation andresuspended in physiologic salt buffer (PBS) as described (Hegde andLingappa, (1996) Cell 85, 217-228). Proteolysis with 0.5 mg/ml PK wasfor 45-90 minutes at 0°. Reactions were terminated with 12.5 mM PMSF andtransferred into 10 volumes of 1% SDS at 100°.

Targeting Studies

In FIG. 2B, translation in the absence of membranes was initiated at32°, following a 30 second pre-incubation in the absence of transcript.Five minutes after transfer to 32°, aurintricarboxylic acid (ATA--Sigma)was added to 75 μM. Aliquots were removed to ice at staggered intervals.Upon collection of the last sample, membranes were added to all but onealiquot. The samples were then returned to 32° for 30 minutes, followedby proteolysis and immunoprecipitation with 3F4. In FIG. 2C, PrPtruncated at StuI was translated at 32° in the presence or absence ofmicrosomes. Five minutes into this incubation, ATA was added as above.After 30 minutes of translation, samples were removed to ice, membraneswere added to the samples lacking them, and incubation at 32° wascontinued for 30 minutes. After translation was completed, chains werereleased with 10 mM EDTA at 26° for 10 minutes. Proteolysis andimmunoprecipitation followed.

Cross-Linking

For samples to be cross-linked, translation products were sedimented andresuspended as above, and divided into equal aliquots. One aliquot wasset aside, and to the other DSS was added to 1 mM and the sample wasincubated at room temperature for 30 minutes. Reactions were terminatedby the addition of 50 mM Tris (pH 8.0), 10 mM EDTA, and 10 μg/ml RNase A(Sigma, St. Louis, Mo.). Where the isolation of lumenal cross-links wasdesired, 0.5% saponin was included as well, followed by sedimentationfor 10 minutes at 75,000 rpm, 4°, in a TLA100. For immunoprecipitationof cross-linked material, saponin was added as above, and antibody wasadded directly to the quenched cross-linking reaction.

Microsomal Membrane Fractionation.

Briefly, rough microsomal membranes (RMs) are prepared as previouslydescribed (Walter and Blobel Methods Enzymol (1983) 96:84-93). Followingthe extraction of lumenal and peripheral membrane proteins, a subset ofthe integral membrane proteins are solubilized using detergent andfractionated by a combination of lectin affinity and ion-exchangechromatography. Individual fractions are reconstituted by removal ofdetergent in the presence of lipids, and the proteoliposomes that formare collected and used to assay for substrate-specific activities. Thefollowing is the detailed protocol for the preparation andcharacterization of an initial set of fractions that demonstrate theprinciples involved. This initial procedure may be modified in severalways as detailed in section [d] below.

Fractionation protocol: i) RMs are prepared according to previouslypublished protocols (Walter and Blobel (1983) supra), Briefly,pancreatic tissue from a recently deceased dog (or pig) is homogenized,and after a centrifugation step to remove debris, nuclei and largesubcellular structures, the remaining material is subject to high-speedcentrifugation. Sedimented material is resuspended and stored frozen at−80° C. All subsequent procedures are carried out either on ice or in acold room at 4° C., unless otherwise noted. ii) The RMs (in 50 mMtriethanolamine-acetate, pH 7.4, 250 mM sucrose, 1 mM DTT) are dilutedto a final concentration of 0.5 equivalents per μl with a buffercontaining 50 mM Hepes, pH 7.4, 250 mM sucrose. Saponin is added to afinal concentration of 0.5% from a 20% stock solution. After thesolution is mixed gently, but thoroughly, the microsomes are isolated bycentrifugation at 100,000 rpm for 10 minutes in a TL100.3 rotor (Beckmaninstruments). The pellet is resuspended at a concentration of 0.5equivalents per μl in buffer containing 500 mM K-acetate, 50 mM Hepes,10 mM EDTA, 125 mM sucrose. The microsomes are again isolated bycentrifugation (100,000 rpm for 20 min in TL100.3 rotor) and resuspendedat 1 equivalent per μl in extraction buffer [350 mM K-acetate, 50 mMHepes, pH 7.4, 5 mM MgCl₂, 15% w/v glycerol, 5 mM 2-mercaptoethanol,EDTA-free protease inhibitors (Roche Molecular Biochemicals) and 0.8%deoxy-BigCHAP (Calbiochem)]. Following extraction for 10 minutes on ice,insoluble material is sedimented at 100,000 rpm for 30 minutes in theTL100.3 rotor. The supernatant is termed the DBC extract, and used insubsequent steps. iii) The DBC extract is incubated with one-fifth toone-seventh volume of immobilized Con A (Pharmacia) with constant andgentle mixing for between 10 and 15 hours, at 4° C. The supernatant (ConA-depleted DBC extract) is removed to a separate tube and either savedon ice (for up to 12 hrs) or for extended storage, frozen in liquidnitrogen and kept at −80° C. The Con A beads are washed three times infive to seven volumes of wash buffer [500 mM K-acetate, 50 mM Hepes, pH7.4, 5 mM MgCl2, 15% w/v glycerol, and 0.5% deoxy-BigCHAP], and thebound proteins eluted with 5 volumes of elution buffer [500 mMK-acetate, 50 mM Hepes, pH 7.4, 20 mM EDTA, 15% w/v glycerol, 250 mMmethyl-alpha-D-mannopyrannoside, 2 mM 2-mercaptoethanol and 0.5%deoxy-BigCHAP] by incubation for 2 hours at 25° C. with constant mixing.The eluted material (Con A elutate) is chilled on ice for subsequentmanipulations. iv) The Con A eluate is diluted with 1.5 volumes ofdilution buffer [50 mM Hepes, pH 7.4, 15% w/v glycerol] and divided forincubation with ion-exchange resins. The diluted Con A eluate is addedto one-twentieth volume of either Q-sepharose-Fast Flow orS-sepharose-Fast Flow (both from Pharmacia), and incubated for 1 hourwith constant mixing. The unbound fraction of the Q-sepharose-Fast Flowand S-sepharose-Fast Flow incubations are added to one-twentieth volumeof either S-sepharose-Fast Flow or Q-sepharose-Fast Flow, respectively,and incubated for one hour at 4° C. with constant mixing. The unboundmaterial of this incubation is set aside (Q/S-flow thru. and S/Q-flowthru). The resin from the second incubation (with S-sepharose andQ-sepharose) are washed in 10 volumes of buffer containing [200 mMK-acetate, 50 mM Hepes, pH 7.4, 15% w/v glycerol, 0.5% deoxy-BigCHAP],and subsequently eluted in 4 volumes of the same buffer containing 1000mM K-acetate. These fractions are termed the Q-FT and S-FT,respectively. The resin from the first ion-exchange incubations (withQ-sepharose and S-sepharose) are washed in 10 volumes of buffercontaining [200 mM K-acetate, 50 mM Hepes, pH 7.4, 15% w/v glycerol,0.5% deoxy-BigCHAP], and subsequently eluted in four volumes of the samebuffer containing 500 mM K-acetate. The resin is eluted a second time infour volumes of the same buffer containing 1000 mM K-acetate. Thesefractions are termed Q-500, S-500, Q-1000, and S-1000, respectively. v)The fractions are reconstituted as follows. First, lipids are preparedby mixing 8 mg phosphatidyl choline (PC, from a 10 mg/ml stocksolution), 2 mg phosphatidyl ethanolamine (PE from a 10 mg/ml stocksolution), 10 mg deoxy-BigCHAP (DBC, from a 100 mg/ml stock solution),and DTT added to 10 mM from a 1M stock. The sample is dried under vacuum(ne heat) and resuspended in 500 μl of buffer [50 mM Hepes, pH 7.4, 15%glycerol]. DBC is added from a 100 mg/ml stock solution to bring thefinal concentraion to approx. 20 mg/ml (˜2% w/v). The lipid mixture isfrozen in liquid nitrogen and stored at −80° C. until needed. Forreconstitutions, 100 μl of the DBC extract or ConA depleted DBC extractis mixed with 100 μl of either Con A elution buffer, Con A eluate, S-FT,Q-FT, S-500, Q-500, S-1000, or Q-1000 fractions prepared as describedabove. In addition, 5 μl of the lipid mixture is added and the samplemixed thoroughly before adding the individual samples to 160 mg ofBioBeads SM2 (Biorad). The mixtures are incubated for 12-18 hours at 4°C. with constant mixing. vi) To recover the proteolipososmes, the liquidphases from step (v) are separated from the biobeads and transferred tofresh tubes. These samples are diluted with five volumes of ice-colddistilled water, mixed, and transferred to 1.3 ml centrifuge tubes onice. The samples are centrifuged for 20 min in TL100.3 rotor (withadaptors for the 1.3 ml tubes) at 70,000 rpm. The supernatant isdiscarded and the pelleted proteoliposomes are resuspended in 30 μl of abuffer containing 100 mM K-Acetate, 50 mM Hepes, pH 7.4, 250 mM sucrose.These are frozen in liquid nitrogen and stored at −80 until used inassays for translocation. vii) Translocation assays are performed asdescribed previously (ref.) using 1 μl of the proteoliposomes from step(vi) per 10 μl of translation reaction.

Purification of PDIp

20,000 equivalents (see Walter and Blobel, 1983, for definition) ofcanine pancreatic rough microsomal membranes were adjusted to 80 mls in50 mM triethanolamine, 250 mM sucrose, 0.2 mM PMSF, 5 μg/ml aprotinin,10 μg/ml chymostatin, 1 μg/ml E64, 5 μg/ml antipain, 1 mM DTT. Withconstant mixing, purified saponin was added slowly to 1% w/v finalconcentration, and after incubation at 4° C. for 15 minutes, membraneswere sedimented by centrifugation for 2 h at 70,000 rpm in 70.1 Ti rotor(Beckman). The supernatants were pooled and applied to a 5 ml column ofConA sepharose at a flow rate of 6.2 mls/hr. The column was washed at 15ml/hr with 25 mls of the above buffer containing 100 mM KAc, and anadditional 25 mls with the above buffer without KAc. The column waseluted at 3 ml/hr at room temperature with 20 mls of the above buffercontaining 1 M α-methyl-mannopyrannoside. The eluate was collected onice and applied at 4° C. to a 1 ml Q-sepharose fast flow column. Thecolumn was washed with 4 ml of 50 mM Hepes, pH 7.5, 2 mM MgAc, 1 mMCaCl2, 1 mM DTT and eluted with 2 mls of this same buffer containing 1 MKAc. The eluate was further fractionated on a Superdex PG 16/16 column(Pharmacia) and 80 1.5 ml fractions collected. 15 μl each of fractions31-56 were analyzed by SDS-PAGE and coomassie staining (FIG. 4 b). Peakfractions containing gp65 were pooled and aliquots used for subsequentsequence analysis (by ProSeq, Salem, Mass.).

Preparation of TRAM-Reconstituted Membranes

Glycoprotein-depleted membranes were prepared as described (Hegde et al,1998c). Purified TRAM, prepared as described (Gorlich and Rapoport,1993), was added at 4× the level present in starting membranes (asjudged by immunoblotting) to the glycoprotein-depleted extract.

Miscellaneous

SDS-PAGE was performed using either 15% gels or 12.5% or 15%Tris/Tricine gels. Bands were either visualized directly byautoradiography, or with the aid of a TranScreen LE intensifying screen(Kodak; Rochester, N.Y.). Quantitation was performed by scanning on anAGFA ArcusII flatbed scanner and densitometry using Adobe Photoshopsoftware. Immunoprecipitations were as described (Chuck and Lingappa,1992)

While all three proteins were efficiently targeted and translocatedacross the ER, they differed dramatically in their topological outcomes.Consistent with previous findings, PrP was synthesized predominantly inthe ^(sec)PrP and ^(Ntm)PrP topological forms, with a small yetsignificant amount of ^(Ctm)PrP (˜7%; FIG. 1C). By contrast, Prl-PrP wassynthesized predominantly in the ^(sec)PrP form, followed by lesseramounts of ^(Ntm)PrP, and essentially undetectable amounts of ^(Ctm)PrP(<2%). Conversely, the topology of βL-PrP was dramatically shiftedtoward ^(Ctm)Prp (˜34%) largely at the expense of ^(sec)PrP. Theseresults suggest that different signal sequences encode information whichdramatically affects subsequent topological events. This informationappears to be in addition to and dependent on the basic targetingfeature common to all three signal sequences. Thus, a chimeric proteinconsisting of the N-terminal twenty amino acids of the cytosolic proteinglobin fused to mature PrP fails to translocate in any of thetopological forms (data not shown). Taken together, the experiments inFIG. 1 demonstrate that the signal sequence of PrP plays a role intopological regulation.

In principle, the influence of the signal sequence on topology describedabove could be ascribed to the well established role of signal sequencesin targeting. If the different signal sequences were to target to the ERat different rates, then the N-terminus of the mature substrate would-besynthesized to varying lengths by the time the ribosome-nascent chaincomplex interacts with the translocon. One consequence of increasingamounts of synthesis during the targeting step may be to favor synthesisof one of the topological forms of PrP (most likely ^(Ctm)Prp, theN-terminal domain of which is not translocated). This hypothesis impliesthat manipulation of the kinetics of PrP targeting should recapitulatethe shifts in topology seen by replacements of the signal sequence. Thispossibility was tested in three ways.

First, the amount of ER-derived canine pancreatic microsomes present inthe translation reactions was titrated and the effect on PrP topologywas examined. As the total concentration of the translocation machinerydecreases, the time between initiation of synthesis and interaction withthe translocon should increase, allowing increasing amounts of thenascent chain to be synthesized prior to targeting. We found thatvarying the concentration of microsomes over a 10-fold range did notimpact the relative ratios of the different topological forms of PrP(FIG. 2A). The decrease in translocation efficiency with decreasingmicrosome concentration, reflected by a decrease in efficiency of signalcleavage, is likely due to the loss of “translocational competence,” therelatively brief period in nascent chain growth when theribosome-nascent chain complex is able to interact productively with thetranslocation machinery (Perara et al. Science (1986) 232:348-352). Asimilar loss of translocation efficiency was observed with othersubstrates (data not shown).

In a second approach, translation reactions were synchronized byinhibiting initiation after 5 min, and microsomes were added atsuccessively later times. We found that regardless of the duration forwhich translation was allowed to occur prior to the addition ofmicrosomes, the percentage of ^(Ctm)PrP synthesized remained constant(FIG. 2B). Again the efficiency of signal cleavage decreased at thelater time points due to an increasing percentage of nascent chains thathad presumably lost translocational competence.

Finally, the targeting step itself was synchronized. In theseexperiments, PrP mRNA was truncated at a defined length and translatedin the absence of microsomal membranes. The resultingribosome-associated nascent chains remain competent for translocation inthe absence of further translation, and can be targeted synchronously bysubsequent addition of microsomes (Perara et al., 1986, supra).Following targeting, the ribosomes were disassembled, and the topologyachieved by the truncated PrP chain, which lacks only a small portion ofthe PrP C-terminus, was compared to the same truncated product that wastargeted co-translationally. We found that synchronizing the targetingof this 223-mer of PrP did not affect the relative ratios of thetopological forms observed (FIG. 2C). Similar results were obtained fora 180-mer as well (data not shown). Taken together, these experimentscollectively argue that the manipulation of the kinetics of thetargeting step of PrP is insufficient to alter the final topology thatis achieved. We infer from these findings that the topology-specificinformation encoded within a signal sequence is likely to act at a stepbeyond targeting.

Since differences in the kinetics of targeting cannot account for theinfluence of the signal sequence on PrP topology, we turned to the othersite of signal sequence action, the ER membrane. Signal sequencerecognition events at the ER membrane appear to occur early duringtranslocation and precede the establishment of a tight ribosome-membranejunction. If the preprolactin and pre-bata-lactamse signal sequencesencode innate differences in the mechanism of their recognition by thetranslocation machinery, these differences might be manifested in thestate of the junction.

Short translocation intermediates of preprolactin and pre-beta-lactamasewere analyzed for their accessibility to proteinase K (PK), a probe forthe state of the ribosome-membrane junction. Consistent with previousreports (Connolly et al., J. Cell Biol (1989) 108:299-307; Jungnickeland Rapoport, Cell (1995) 82:261-270), the tight seal of the junctionshielded the preprolactin 86-mer from proteolytic attack. By contrast,the pre-beta-lactamase 84-mer was largely susceptible to digestion,indicative of an open ribosome-membrane junction (FIG. 3A). To determineif this difference was attributable to signal sequence function, weexchanged the signal sequences of preprolactin and pre-beta-lactamase(constructs βL-Prl and Prl-βL). We found that the βL-Prl 81-mer wasaccessible to protease digestion while the Prl-βL 93-mer was not (FIG.3A). Similar results were obtained with translocation intermediatesapproximately 20 amino acids longer (data not shown). These data arguethat signal sequences can differentially influence the state of theribosome-membrane junction during the early biogenesis of secretoryproteins.

Given the above results, modulation of the ribosome-membrane junction bythe signal sequence seemed to be a plausible mechanism for theregulation of PrP topology. If so, the topological form of PrP with itsN-terminus in the cytosol (^(Ctm)PrP) may be expected to arise fromchains that did not establish a tight ribosome-membrane junction earlyin translocation. To test this hypothesis, increasingly longertranslocation intermediates of PrP, Prl-PrP, and βL-PrP were assembledat the ER and separated from non-targeted chains by sedimentation, andthe state of the ribosome-membrane junction was analyzed by PK digestion(FIG. 3B).

When truncated at a point corresponding to a 61-mer of wild-type PrP,all three substrates are fully accessible to protease, and thus havefailed to establish a tight junction. However, the three constructs showdifferential protease accessibility at a translocation intermediate only52 amino acids longer, even before the synthesis of the transmembranedomain. Prl-PrP, which makes very little ^(Ctm)PrP, is largely shieldedfrom digestion at this point, while a greater percentage of βL-PrPchains are accessible to the protease. At this truncation point, allthree constructs are resistant to extraction from the membrane with highsalt (0.5 M potassium acetate; data not shown), suggesting that theribosome is stably associated with the translocation channel.Furthermore, βL-PrP nascent chains remain more accessible to PK thanPrl-PrP chains throughout their biogenesis. Most of the unprotectedchains retain their signal sequences, likely as a consequence of theopen junction. The observed diminishment of protease protection in allthree substrates with increasing chain length probably reflectsjunctional opening during the synthesis of ^(Ntm)PrP. Collectively,these results argue that signal sequence-mediated regulation of theribosome-membrane junction can have dramatic consequences for subsequenttranslocational events.

The putative role of the ribosome-membrane junction in governing finaltopology suggests that trans-acting factors which regulate the junctioncould influence PrP biogenesis. The TRAM glycoprotein is thought tointeract with signal sequences to facilitate the initial associationbetween the ribosome-nascent chain complex and the translocon (Voigt etal., J. Cell Biol (1996) 134:25-35). The signal sequences of somesubstrates (such as preprolactin) do not depend on TRAM fortranslocation, though most (including pre-beta-lactamase) areTRAM-dependent (Gorlich et al., Nature (1992) 357:47-52; Gorlich andRapoport, Cell (1993) 75:615-630; Voigt et al., 1996, supra). AlthoughTRAM has additionally been implicated in regulating theribosome-membrane junction during the translocation of other substrates(Hegde et al., Cell (1998) 92:621-631), functional studies have thus farfailed to demonstrate a direct or critical role for TRAM in PrP topology(Hegde et al., Mol. Cell (1998) 2:85-91). Thus, we reasoned that itshould be possible to dissociate the TRAM-dependence feature of a signalsequence from its role in PrP topology.

We sought to identify mutations within the PrP signal sequence thatdifferentially modify topology but not dependence on TRAM. By analogy tothe effects of charge on the topology of signal-anchor proteins (e.g.,Sipos and von Heijne, Eur J. Biochem (1993) 213:1333-1340; Spiess, FEBSLett (1995) 369:76-79), we reasoned that non-conservative mutations inthe PrP signal sequence may affect PrP biogenesis. Leaving thehydrophobic core of the signal sequence intact to allow efficienttargeting, we replaced either codons 2 and 3 or codons 4 and 5 witharginine or aspartic acid codons [PrP_((R2,3)), PrP_((D2,3)),PrP_((R4,5)) and PrP_((D4,5)), respectively; see FIG. 4A). Topologicalanalysis of PrP_((R2,3)) and PrP_((R4,5)) revealed a decrease in^(Ctm)PrP synthesis while PrP_((D2,3)) and PrP_((D4,5)) both showedincreased ^(Ctm)PrP synthesis (FIG. 4B).

To assess the TRAM dependence of these mutant signal sequences we fuisedeach of them to preprolactin in place of the native preprolactin signalsequence. The ability of these constructs to translocate intoglycoprotein-depleted proteoliposomes either containing or lackingpurified TRAM was measured. TRAM-dependence was defined as thepercentage of chains that require TRAM to translocate. We found that allfour mutated signal sequences were less TRAM-dependent than thewild-type PrP signal sequence, despite their divergent effects on PrPtopology (FIG. 4C). Thus, the TRAM-dependence of the PrP signal sequencecan be dissociated from the role of the signal sequence in regulatingtopology, arguing for a previously unappreciated mechanism by whichsignal sequences influence topology.

Mutations in the membrane-spanning domain can influence the topology ofPrP (Hegde et al., Science (1998) 279:827-834). Because transmembrane(TM) domains, like signal sequences, can elicit changes in theribosome-membrane junction (Liao et al., Cell (1997) 90:31-41), it isplausible that the mechanisms by which these two domains regulatetopology are related. Just as certain signal sequences open theribosome-membrane junction, the TM domain of PrP may be capable ofeliciting an opening of the junction. In this scenario, TM domainmutants that favor ^(Ctm)PrP would elicit a greater opening of thejunction than wild-type PrP, while mutants-that favor ^(sec)PrP wouldact to maintain a closed junction. We tested this hypothesis byanalyzing PrP constructs that contained various combinations of changesin both the signal sequence and TM domains.

Replacement of conserved alanines in the TM domain with valines markedlyincreases the proportion of PrP made in the ^(Ctm)PrP topology (Hegde etal., Science (1998) 279:827-834). One of these mutants [PrP_((Av3))which generates approximately 40% of the molecules in the ^(Ctm)PrPform] was engineered with either a preprolactin or pre-beta-lactamasesignal sequence [Prl-PrP_((Av3)) and βL-PrP_((Av3))]. If the AV3mutation causes the ribosome-membrane junction to open independently ofthe signal sequence, then the Prl-PrP_((AV3)) construct shouldsynthesize roughly as much ^(Ctm)PrP as PrP_((Av3)). Strikingly, weobserved that Prl-PrP_((Av3)) reduces ^(Ctm)PrP synthesis to barelydetectable levels (FIG. 5A, middle panel). Conversely, βL-PrP_((Av3))makes almost exclusively ^(Ctm)PrP (FIG. 5A, right panel).

Similarly, according to the hypothesis stated above, a TM mutant whichfavors ^(sec)PrP synthesis should cause the ribosome-membrane junctionto close. When we replaced the signal sequence of the TM mutantPrP_((G123P)), which makes only ^(sec)PrP with the preprolactin orpre-beta-lactamase signal sequence [Prl-PrP_((G123P)) andβL-PrP_((G123P))], we found that all three substrates were synthesizedonly as ^(sec)PrP However, closure of the ribosome-membrane junctionshould stimulate overall translocation efficiency, since chainssynthesized with a closed junction have the ER lumen as their onlyoption for exit from the ribosome. Instead, we found thatβL-PrP_((G123P)) translocates relatively poorly compared toPrP_((G123P)) and Prl-PrP_((G123P)). This result argues that the G123Pmutation does not necessitate the production of ^(sec)PrP by closing theribosome-membrane junction, but rather by preventing the translocationof chains which would otherwise become ^(Ctm)PrP.

The findings above suggested that rather than acting independently ofthe signal sequence, the TM domain is constrained by the precedingaction of the signal sequence. To test directly whether the TM domainhas any bearing on the state of the ribosome-membrane junction dictatedby the signal sequence, we analyzed the state of the junction forPrl-PrP_((AV3)) and βL-PrP_((G123P)) at a point after the emergence ofthe TM domain. We found that the junction remains open forβL-PrP_((G123P)) and closed for PrI-PrP_((Av3)), irrespective of theidentity of the TM domain (FIG. 5C). These results suggest that theinitial action of the signal sequence on the ribosome-membrane junction,even before the synthesis of the TM domain, is critical to subsequenttopological determination.

The observation that PrP chains are committed to a transmembranetopology prior to the synthesis of the TM domain was unexpected. Whymight it be important to place the key regulatory steps at such an earlypoint in biogenesis? One possible reason is that certain early events inthe folding of PrP are not readily reversible at a later point inbiogenesis when the TM domain has been synthesized. Thus, it may beimportant to initiate these early folding events in a temporally andspatially restricted manner (given that the N-terminus can ultimatelyreside in one of two environmentally different compartments). To examinethis possibility, we sought to identify interactions between PrP and themachinery of protein folding that are initiated in a topology specificmanner at a time before topology has been formalized.

In initial experiments, we used cross-linking to identify proteins thatdisplayed preferential association with either PrP_((G123P)) orPrP_((AV3)). While several proteins interact comparably with bothsubstrates, a 65 kDa protein (p65) cross-links more strongly toPrP_((G123P)) than to PrP_((AV3)) (FIG. 6A). We then similarly analyzedPrl-PrP and βL-PrP truncated at the same location and found that p65preferentially cross-links to Prl-PrP, suggesting that nascent PrPchains in the process of being made in the ^(sec)PrP topologyspecifically associate with this factor (FIG. 6B). To determine whenduring PrP biogenesis this interaction is initiated, we examined earlytranslocation intermediates of Prl-PrP and βL-PrP for the differentialpresence of this cross-link. Remarkably, we found that p65 and aslightly smaller protein cross-link preferentially to the Prl-PrPtranslocation intermediate truncated at codon 113 of wild-type PrP (FIG.6C). Thus, association of p65 with nascent PrP chains very early inbiogenesis correlates with the propensity of the substrate to ultimatelybe made in the ^(sec)PrP topology.

We took advantage of the biochemical properties of the p65 cross-linkedadduct to purify and identify this protein. The adduct was extractableby saponin, implying a lumenal localization for p65; it migrated as a 4S protein by sucrose gradient analysis, suggesting it was monomeric; itwas retained on a ConA-sepharose column, indicating that p65 isglycosylated; and it was also retained on a Q-sepharose column,suggestive of a negative net charge imparted by p65 on an otherwisenet-positive PrP molecule. When these properties were combined in astepwise fractionation of canine rough microsomes, a single major 65 kDaprotein was purified (FIG. 6D). Sequencing of peptide fragmentsgenerated by cyanogen bromide cleavage of this protein identified it asPDIp, a pancreas-specific member of the Protein Disulfide Isomerase(PDI) family of chaperones (Desilva et al., Cell Biol (1996) 15:9-16;Elliott et al., Eur J. Biochem (1998) 252:372-377).

The identity of p65 as PDIp was confirmed by immunoprecipitation withantibodies raised against PDIp (FIG. 6E). In addition, the slightlysmaller cross-linking partner was immunoprecipitated by antibodies toubiquitous PDI, further arguing for a specific interaction between PrPand this family of proteins. The immunoprecipitation studies confirmedthat both PDI and PDIp cross-link more strongly to Prl-PrP than toβL-PrP (FIG. 6E). This differential crosslinking of ^(sec)PrP favoringconstructs to PDI was also observed in microsomes isolated from brain,the tissue in which PrP is predominantly expressed. In this case, onlycross-links to the ubiquitous PDI were observed, consistent withprevious observations that PDIp is not expressed strongly in braintissue (Desilva et al., 1996, supra).

We next sought to determine if ^(sec)PrP specifically interacts with anyother lumenal proteins early during biogenesis. Examination of thesaponin extractable cross-links revealed prominent interactions betweenthe ^(sec)PrP favoring constructs [PrP_((R2,3)) and PrP_((R4,5))] andproteins of approximately 30 kDa, 60 kDa, and 65 kDa (FIG. 6F). Similarcross-links to the ^(Ctm)PrP favoring mutants [PrP_((D2,3)) andPrP_((D4,5))] were markedly diminished. As expected, the 60 kDa and 65kDa cross-links were identified by immunoprecipitation as PDI and PDIp(data not shown). The identity of the 30 kDa cross-linking partnerremains unknown. These cross-links are not observed simply as afortuitous consequence of the lumenal localization of ^(sec)PrP becauseprominent cross-links to lumenal proteins were not observed with earlytranslocation intermediates of preprolactin (FIG. 6F and data notshown). The cross-linking data collectively argue that one consequenceof signal sequence-mediated events in-early PrP biogenesis is tofacilitate differential interactions between the nascent chain andfactors which may subsequently participate in its biogenesis.

Example 2 Pathogensis of Altered Topology of Prion Protein is Common toGenetic and Infections Prion Diseases

Prion diseases can be infectious, sporadic and genetic (Prusiner, S. B.PNAS. (1998) 95:13363-13383; Weissmann, C J.Biol.Chem (1999) 274:3-6;Johnson New Eng J Med (1998) 339:1994-2004; Horwich Cell (1997)89:499-510). The infectious forms of these diseases, including bovinespongiform encephalopathy and Creutzfeldt-Jakob disease, are usuallycharacterized by the accumulation in brain of the transmissiblepathogen, and abnormally folded insoform of the prion protein (PrP)termed PrP^(Sc.). However, certain inherited PrP mutations appear tocause neurodegeneration in the absence of PrP^(Sc) (Brown, Ann Neur(1994) 35:513-529; Tateishi, J. et al., Neurology, 1990, 40:1578-1581;Tateishi, Neurology, (1996) 46:532-537, Tateishi, J., Brain Pathology,(1995) 5:53-59), instead working by favoured systhesis of ^(Ctm)PrP, atransmembrane form of PrP (Hegde, et al. Science (1998) 279:827-834).Certain mutations in PrP (including the human prion disease-associatedA117v mutation) alter its biogensis at the endoplasmic reticulum (ER),causing a higher percentage of PrP molecues to be synthesized in thetransmembrane form ^(Ctm)PrP. Expression of ^(Ctm)PrP-favouringmutations in transgenic mice resulted in neurodegenerative changessimilar to those observed in prion disease (Hegde, et al. Science (1998)279:827-834) The detection of ^(Ctm)PrP-favouring mutations suggestedtheat elevated ^(Ctm)Prp causes neurodegeneration (Hegde, et al. Science(1998) 279:827-834), but whether this mechanism of neurodegeneration isinvolved in the pathogenesis of transmissible prion disease has beenunclear. To explore this question mice were generated with mutant PrPtransgenes differing in their propensity to form ^(Ctm)PrP, andsubsequently their suceptibility to PrP^(Sc) induced neurodegenerationwas assessed.

Cell-free translation and translocation. Transcription of the relevantcoding regions using SP6 polymerase, translation in rabbit reticulocytelysate containing imcrosomal membranes from dog pancreas, andproteolysis were essentially as described previously (Hegde, et al.Science (1998) 279:827-834). Translation reactions were carried out at32° C. for 40 minutes, and proteolysis reactions at 0° C. for 60 minutesusing 0.5 mg/ml PK. Products were immunoprecipitated with the R073antibody (Rogers, M. et al., J. Immun, 1991, 147:3568-3574), separatedby SDS-PAGE on 15% acrylamide gels, and visualized by autoradiography.TABLE 1 Transgenic mouse production and characterization. TransgenicTransgenic % Ctm in Level of PrP Age spont CtmPrP in Sc237 inc. LineNumber Line Name vitro expr (rel to Sha Ctm-index disease (days) vivotime (days) TgSHaPrp(S F1788 6 4 24 − − 323 +/− TE)H 14(9/9) TgSHaPrPE15781 31 0.4 12 − −  70 +/− 2 (6/6) (A117V)L TgSHaPrP E15727 31 4 124572 +/− 35 +  55 +/− (6/6) (A117V)H (5/5) TgSHaPrP(N E15786 35 1 35 − −311 +/− (3-3) 1081)L TgShaPrp(N E15790 35 5 175 312 +/− + 233 +/− 2(9/9)1081)H 24(7/7) TgShaPrP(K E12485 48 0.4 19 − − 257 +/− 2 H-IIl)L (9/9)TgSHaPrP(K F1220 48 1 48 472 +/− + 181 +/− H-II)M 13(6/6) 5(10/10)TgSHaPrP(K F1198 48 4 192  58 +/− 11 ++ ND^(b) H-II) H (24/24)^(a)Table 1 Characteristics of transgenic mouse lines used in this study.The values for % Ctm in vitro were derived from quantitation of FIG. 8a.The levels of PrP expression were determined by quantitive westernblotting with the 13A5 monoclonal antibody and re rexpressed relative toPrP expression in Syrian hamster*Sha; see FIG. 1B for a representative experiment). The Ctm-index foreach transgenic line is derived by multiplying the values in thepreceeding two columns. ‘Age spont disease’ indicates the age of onsetof clinical symptons [average +/− SEM (n/n.)]. biochemical assay fordetermining the presence of ^(Ctm)Prp in viro was as previouslydescribed^(9,) and carried out on either clincially ill animals (in thecase of transgenic# lines developing illness) or mice over 600 days of age (in the case oftransgenic lines that don't develop neurodegeneration). ‘Sc237 inc.time’ indicates the time from inoculation with Sc237 Sha prions todevelopment of neurologic signs of dysfunction [average +/− SEM (n/n.)].^(a)Data from ref. 9.^(b)ND indicates not determined.were generated as previously described (Manson, et al. Neurodegeneration(1994) 3:331-340 and references therein). PrP expression was assessed byimmnunoblotting of brain tissue homogenate with 13A5 mAb (Kascsak, R. J.et al., J.Virology, 61:3688-3693), comparing to serial dilutions ofnormal Syrian hamster brain tissue (FIG. 8 b and Table 1). Observationof these mice for development of spontaneous illness was as previouslydescribed (Prusiner Ann Neur (1982) 11:353-358). The double trangenicmice expressing both SHaPrP and MoPrP (see FIG. 11) were generated bycrossing Tg[SHaPrP]/Prnp^(0/0) (line A3922)⁹ to Tg [MoPrP]/Prnp^(0/0)(line B4053) (Telling Genes Dev (1996) 10:1736-1750). Transmissibility(see FIG. 10) was assessed by intracerebral inoculation of 1% brainhomogenate (w/v) into mice (30 μl per animal) or hamsters (50 μl peranimal) as previously described (Prusiner Ann Neur (1982) 11:353-358).the Sc237 strain of hamster prions (used in FIG. 9) and RML strain ofmouse prions (used in FIG. 11) have been described previously (Marsh JInfect Dis (1975) 131:104-110; Chandler Lancet (1961) 1:1378-1379).

Assessment of brain for ^(Ctm)Prp and PrP^(Sc.) Brain tissue (eitherfreshly removed or stored frozen at −80° C. following flash-freezing inliquid nitrogen) was homogenized in PBS (at 5% w/v or 10% w/v) bysuccessive passage through 16, 18 and 20 gauge needles. For ^(Ctm)PrPdetection (‘mild’ proteolysis conditions), 17 μl aliquots of the sample(at a concentration of 25 μg/μl) were adjusted (in a final volume of 20μl) to 1% NP-40, 0.25 mg/mk PK and incubated for 60 min on ice. ForPrP^(Sc) detection (‘harsh’ proteolysis conditions), 17 μl samples (at aconcentration of 25, μg/μl) were adjusted (in a final volume of 20 μl)to 0.5% NP-40, 0.5% deoxycholate, 0.1 mg/ml PK and incubated for 60minutes at 37° C. It should be noted that the difference between mildversus harsh digestion conditions, while operational, is not subtle, asit involves a 37° change in temperature of incubation, and the presenceof non-ionic detergent versus mixed micelles of non-ionic and ionicdetergents. The proteolysis reactions were terminated by the addition ofPMSF to 5 mM, incubating an additional 5 minutes, and transferring thesample to 5 volumes of boiling 1% SDS, 0.1M Tris, pH 8.9. Samples werethen digested with PNGase as directed by the manufacturer, resolved by10% tricine-SDS-PAGE, transferred to introcellulose, and probed witheither the 3F4 or 13A5 monoclonal anitbody (Kascsak J Virol61:3688-3693), or the RO73 polyclonal anitbody (Rogers J Immunol (1991)147:3568-3574).

Results

Shown in FIG. 8 a are in vitro translocation products of four mutants ofSyrian hamster (Sha) PrP that alter the amount of ^(Ctm)PrP synthesizedat the ER. Transgenic mice expressing each of these mutant PrPs in theFVB/Prnp^(0/0) background were generated and characterized (see Table 1and FIG. 8 b). The Tg[SHaPrP(KH→II)_(H)], Tg[SHaPrP(KH→II_(m)],Tg[SHaPrP(A117V)_(H)] and Tg{SHaPrP(N108I)_(H)] mice were observed todevelop signs and symptons of neurodegenerative disease at approximately60, 472, 572 and 312 days, respectively (FIG. 8 c and Table 1). Bycontrast, neither the Tg[SHaPrP(ΔSTE)] mice nor mice expressing lowerlevels of the disease-associated transgenes {Tg[SHaPrP(KH→II)_(L)],Tg[SHaPrP(A117V)_(L)] and Tg[SHaPrP(N108I)_(L)]} developed spontaneousdisease (Table 1 and data not shown). biochemical analyses of braintissue from each of these lines of transgenic mice revealed elevated^(Ctm)PrP, but not PrP^(Sc), in the lines which developed disease (FIG.8 d). Together, the data in FIG. 8 recapitulate the point that increasedsynthesis of the ^(Ctm)PrP form of PrP is associated with thedevelopment of neurodegenerative disease.

More remarkable is the apparent dose response, seen in two ways, between^(Ctm)PrP and severity of disease. First, the more strongly that^(Ctm)PrP systhesis is favoured at the ER (KH→II>N108I>A117V), theearlier the onset of spontaneous disease (Table 1). Second, lowering thelevel of expression of each of these mutations below an apparentthreshold abrogates both the generation of ^(Ctm)PrP (FIG. 8 d and ref.9) and development of disease (Table 1). Furthermore, the threetransgenic lines expressing the KH→II mutation develop disease at timesinversely correlated with their respective levels of expression (Table1). These observations demonstrate that bot the ^(Ctm)PrP-favouringquality of a mutation and its level of expression contribute to thedevelopment of neuodegeneration.

This panel of transgenic mice with differing propensities to make^(Ctm)PrP was used to dissect the relationship between ^(Ctm)PrP andPrP^(Sc). We first examined the susceptibility to PrP^(Sc) of transgenicmice with identical levels of transgene expression but differingpropensities to make ^(Ctm)PrP: Tg[SHaPrP(□STE)] andTg[SHaPrP(A117V)_(H)]. Upon inoculation with Sc237 hamster prions, wefound that the Tg[SHaPrP(□STE)] and Tg[SHaPrP(A117V)_(H)] mice developedillness at aproximately 323 and 54 days, respectively (FIG. 9 a, Table1). Biochemical analysis of representative mice at the time of diseaseonset revealed that the Tg[SHaPrP(□STE)] mice contained substantiallymore PrP^(Sc) than the Tg[SHaPrP(A117V)_(H)] mice (FIG. 9 b). Thus, thetransgenic line that generates higher CtmPrP is more susceptible toPrP^(Sc), developing disease at a lower level of overall PrP^(Sc)accumulation.

We next compared the susceptibility to Sc237 of Tg[SHaPrP(KH→II)_(L)]versus Tg[SHaPrP(A117V)_(H)] (FIG. 9 e). By keeping the mutationconstant, issues regarding a potential barrier to propagation areavoided, while still changing the propensity to generate CtmPrP bymodulating level of expression. As expected, lowering the level ofexpression increased the incubation time to disease following 5c237inoculation. More remarkably however, we found that with both the KH→IIand A117V mutants, the lower level expressor contained the higher levelof PrPSC at the time of disease onset (FIG. 9 d,f). A similar inverserelationship between level of expression and amount of PrPSC at diseasehas been observed with mice expressing different levels of wild type PrP(ref.10). Thus, as above, the mice with a diminished propensity to formOtmPrP had accumulated higher levels of PrPSC at onset of disease. Tointegrate the above inoculation data into a single plot, we ranked thedifferent lines of transgenic mice by their relative propensities togenerate CtmPrP using a measure we term the Ctm-index (see Table 1).This index is derived by multiplying the percent of chains synthesizedin the CtmPrP topology by the level of transgene expression, thusincorporating the two parameters known to influence CtmPrP generation.FIG. 9 g shows that a clear relationship exists between the Ctm-indexand the amount of PrPSC that had accumulated at disease onset. Thisrelationship suggests that the ability of PrPSC to cause disease is afunction of the propensity of the host to generate CtmPrP.

The data in FIG. 9 demonstrate two important points. First, verydifferent levels of PrP^(SC) accumulation are observed at the time ofonset of clinical disease upon inoculation of the various lines oftransgenic mice. Given that the strain of mice is identical in eachcase, with the only differences being in the nature and level ofexpression of the PrP transgene, this observation underscores theconclusion that accumulation of protease-resistant PrP^(Sc) is notlikely to be the most proximate cause of disease; subsequent events(apparently involving ^(Ctm)PrP) are likely to be involved. Second,there is a relationship between the amount of accumulated PrP^(SC) andthe factors that modulate ^(Ctm)PrP generation. Such a relationshipargues that ^(Ctm)PrP and PrP^(SC) are part of a pathway in which theypotentially can influence, either directly or indirectly, each other'smetabolism.

One way to reconcile the data in FIG. 9 g is if accumulation of PrP^(SC)caused increased generation of ^(Ctm)PrP which then elicitedneurodegeneration. Thus, transgenic mice that have an elevatedpropensity to generate ^(Ctm)PrP (i.e., a high Ctm-index) would requireless PrP^(SC) accumulation before ^(Ctm)PrP generation is increasedbeyond the threshold needed to cause disease. This model would explainthe inverse relationship observed in FIG. 9 g, and makes two additionalpredictions. First, since transgenic mice that substantially favoursynthesis of PrP in the ^(Ctm)PrP form can entirely circumvent therequirement for PrP^(SC) in the developement of neurodegenerativedisease, tissue from such mice should not be infectious. Second,^(Ctm)PrP levels should increase during the course of PrP^(SC)accumulation in infectious prion disease. These predictions were tested.

To assess the transmissiblity of ^(Ctm)PrP-associated disease, brainhomogenate from clinically ill Tg[SHaPrP(KH→II)_(H)] mice wereinoculated intracerebrally into four hosts: i) Tg[SHaPrP(KH→II)_(L)]mice expressing the KH→II mutation at low levels, ii) Tg[SHaPrP] miceoverexpressing wild type SHaPrP, iii) FVB/Prnp^(0/0) mice with ahomozygous disruption of the PrP gene, and iv) Syrian hamsters. As shownin FIG. 10, homogenate from terminally sick Tg[SHaPrP(KH→II)_(H)] micedid not induce neurological illness at rates different than controlTg[SHaPrP] homogenate when directly compared in three independent hosts.Furthermore, biochemical and pathological examination of representativebrain tissue from FIG. 10 at up to 625 days after inoculation did notshow any evidence of PrP^(Sc) or neurologic disease in eitherexperimental or control animals (data not shown). Yet, inoculation ofTg[SHaPrP(KH→II)_(L)] mice with Sc237 prions readily generated PrP_(SC)in brain (FIG. 2 d), which re-transmitted disease toTg[SHaPrP(KH→II)_(L)] and Tg[SHaPrP] mice (data not shown). Thus,although PrP(KH→II) is capable of being formed into PrP^(SC), the^(Ctm)PrP. associated disease in Tg[SHaPrP(KH→II)_(H)] mice does notgenerate detectable PrP^(SC) and therefore is not infectious. Lack oftransmission provides further support for the hypothesis thatneurodegeneration in these genetic prion diseases is caused by ^(Ctm)PrPdirectly. The second prediction made on the basis of the data in FIG. 9g was that accumulation of PrP^(SC) in infectious prion disease shouldinduce the increased generation of ^(Ctm)PrP, which would subsequentlylead to neurodegeneration. Unfortunately, testing this predictiondirectly is hampered by the biochemical properties of the accumulatingPrP^(SC) (Meyer PNAS (1986) 83:2310-2314). Being highlyprotease-resistant and heterogeneous in its fractionation, it tends tocontaminate substantially all subcellular fractions. Additionally, itinterferes with the assays for ^(Ctm)PrP. detection, which is also basedon protection from proteases. Because PrP^(SC) is not readily degradedby the cell and accumulates to very high levels¹², even very smallamounts of contamination of suboellular fractions are sufficient to makedetection of subtle increases in ^(Ctm)PrP difficult. Thus, an alternatemethod is required to monitor the effect of accumulating PrP^(SC) on thetopology of newly synthesized PrP (see FIG. 11 a).

In order to design such an experiment, we took advantage of threeobservations. First, a species barrier to PrP^(SC) conversion existsbetween mouse and Syrian hamster (Prusiner Cell (1990) 63:673-686;Pattison Res Vet Sci (1968) 9:408-410). Second, in contrast to thespecies barrier for PrP^(SC) formation, no species specific differencesin the synthesis, translocation or topology are observed between mousePrP (MoPrP) and SHaPrP (Hegde Science (1998) 279:827-834). And finally,monoclonal antibodies highly specific to SHaPrP (which do not crossreact with MoPrP) are available to distinguish between expression ofthese two PrP transgenes¹⁵. Thus, in such double transgenic animals wecan use hamster ^(Ctm)PrP formation as a ‘reporter’ during the course ofaccumulation of mouse PrP^(SC). For this experiment, the doubletransgenic mice which synthesize both MoPrP and SHaPrP are inoculatedwith mouse prions (of the RML strain). Then, at various intervals duringthe time course of accumulation of PrP^(SC) and development of disease,individual mice are sacrificed and examined for total PrP^(Sc)accumulation and for the presence of hamster ^(Ctm)PrP (see FIG. 4 a).The principle is that following inoculation, only MoPrP will be asubstrate for prion replication and PrP^(SC) formation¹³. The effect ofthis PrP^(Sc) accumulation on the ability of cells to generate (or notgenerate) ^(Ctm)mPrP can be assessed by examining SHaPrP.

Clinical disease was noted to develop in these animals approximately 9weeks after inoculation (data not shown). We found that PrP^(SC)accumulated in these mice during this 9 week time course, with theearliest detectable times being approximately 5-6 weeks (FIG. 11 b). Asexpected, the SHaPrP was not noted to have formed any PrP^(SC) by bothbiochemical criteria in this study (FIG. 11 b) and infectivity criteriain prior studies¹³. Remarkably however, a significant increase in theamount of ^(Ctm)PrP was noted upon examination of the SHaPrP (FIG. 11c). Such an increase was not observed in a parallel set of mice that didnot receive the inoculum (data not shown). These findings, coupled withthe observation that ^(Ctm)PrP is capable of causing neurodegenerationin the absence of an transmissible forms of PrP (ref. 9, FIGS. 8 and.10), suggest that PrP^(Sc) accumulation may cause disease by inducingthe synthesis of ^(Ctm)PrP de novo.

The findings described herein suggest causal relationships betweenPrP^(SC) accumulation, the events of ^(Ctm)PrP formation and metabolism,and the development of neurodegenerative disease. Three complementaryand independent lines of evidence argue for this conclusion. First,increasing the generation of ^(Ctm)PrP beyond a certain threshold (bymodulating a combination of PrP mutation and level of expression)results in neurodegeneration in the absence of PrP^(SC) formation (FIG.8, FIG. 10 and ref. 9). Second, the amount of accumulated PrP^(SC)needed to cause neurodegenerative disease is influenced by thepropensity of the host to generate ^(Ctm)PrP (FIG. 9). And third, thebrain appears to contain increasing levels of ^(Ctm)PrP during thecourse of accumulation of PrP^(SC) (FIG. 11). Taken together, the dataare suggestive of three successive stages in the pathogenesis of priondiseases (FIG. 12).

Infectious prion diseases are proposed to work by initiating the stepsof Stage I, the accumulation of PrP^(SC). Genetic prion diseases couldin principle work at either Stage I or II. If the PrP mutation inquestion results in the spontaneous formation of PrP^(SC), Stage I wouldbe initiated, PrP^(SC) would replicate and accumulate, and subsequentlycause increased elevation of ^(Ctm)PrP (stage II). Such a mechanismseems plausible for the E200K mutation thought to cause certain geneticvariants of Creutzfeldt-Jakob disease¹⁶. Thus, PrP^(Sc) is seen in thesepatients' 17, and the disease is readily transmissible to experimentalanimals⁷. Alternatively, certain other PrP mutations could bypass stageI altogether by directly causing an increase in ^(Ctm)PrP generation.The A117V mutation resulting in human Gerstmann-Straussler-Scheinker¹⁸disease is likely to work by such a mechanism. This would explain whythis disease has not been transmissible⁶⁻⁸, and why PrP^(SC) has notbeen detected in these patients' brain tissue^(6,9).

The final stage in prion disease pathogenesis includes the mechanisms bywhich ^(Ctm)PrP, once generated, leads to neurodegenerative disease. Themechanism by which this occurs and the intracellular pathways that areinvolved remain entirely unclear. However, it does not appear to be thecase that ^(Ctm)PrP is simply misfolded, retained or accumulated in theER, or eliciting an unfolded protein response. This is suggested by theobservation that essentially all of the ^(Ctm)PrP has been traffickedbeyond the ER⁹, the site of the presently known quality controlmachinery for protein folding in the secretory pathway^(19,20).Additionally, disease can be elicited by transgenes expressed at closeto physiologic levels, as is the case with Tg[SHaPrP(KH→II)_(M)] animalsor human cases of GSS containing the A117V mutation. Thus, a moreselective means by which ^(Ctm)PrP induces neurodegeneration issuggested by the available data.

The framework described in FIG. 12 suggests several new avenues forfuture studies. First, the regulated events in^(Ctm)PrP biogenesis andtrafficking remain to be elucidated. The reconstitution of the earlyevents of PrP translocation and topology determination in a cell-freesystem amenable to fractionation appears to be a promising avenue forthe identification of trans-acting factors regulating ^(Ctm)PrPsynthesis^(21,22). Additionally, the availability of several mutantsinfluencing topology should facilitate studies aimed at defining laterevents in the trafficking of ^(Ctm)PrP. Second, insights gained fromstudying the metabolism of ^(Ctm)PrP will undoubtedly allow for a betterunderstanding of how these events are modulated in trans by PrP^(SC)accumulation. Such studies are likely to better delineate therelationship between the events of PrP^(SC) accumulation and ^(Ctm)PrPmediated neurodegeneration. Given that PrP^(SC) accumulation may impactupon several metabolic functions of the cell^(23,24,25), it is plausiblethat one or more of these influence ^(Ctm)PrP generation to elicitdisease. The availability of PrP mutants that act at a step beyondPrP^(SC) accumulation to directly cause ^(Ctm)PrP mediatedneurodegeneration (ref. 9 and FIG. 8) should facilitate the dissectionof the downstream events in prion disease pathogenesis.

Example 3 Effect of Prolactin, Beta Lactamase, and Immunoglobulin GHeavy Chain Signal Sequences on Protein Conformation

In general, a dichotomy has been accepted between a protein beingproperly folded versus misfolded (Ellgaard et al. (1999) Science 286;1882-1888). In other words, the possibility that proteins might havemore than one properly folded state and might even be able to select oneversus another folded state at different times or circumstances has notbeen considered. Because a linear polypeptide sequence folding indifferent ways could have substantially different shapes, physicalproperties and biological activities, this new level of regulationgreatly increases the information content of the genome and thepotential for regulation of gene expression available to the cell. Totest this hypothesis we first selected three unrelated, simple secretoryproteins, namely, prolactin (prl), beta lactamase (βlac), andImmunoglobulin G heavy chain (IgG), to determine whether the individualsignal sequences would be equally efficient at translocation of theirown passenger sequences across the ER membrane (see FIG. 13) as those ofdifferent nascent chains.

We truncated aliquots of these cDNAs at each of three unique restrictionendonuclease recognition sites, so that when transcribed the truncatedcDNAs generated RNA transcripts encoding longer and longerC-terminally-extended lengths of these secretory proteins, and finallyexpressed these transcripts in cell-free translation systemssupplemented with microsomal membranes. As can be seen in FIG. 14,despite the fact that these three signal sequences are equally highlyefficient at translocating their own nascent chains, radically differentribosome-membrane junctions are established for translocation ofdifferent nascent chains, as determined by accessibility of the nascenttruncated chain to digestion with proteinase K (see cartoon to right ofeach panel of FIG. 14).

Initially upon targeting to the ER membrane, all three chains remainaccessible to protease (left hand panels of FIG. 14A-C). Shortlythereafter, in the case of prolactin, a tight seal is established as thenascent chains is translocated directly to the ER lumen (see middlepanel of FIG. 14A). However, in the case of both beta lactamase and IgGheavy chain, the ribosome-membrane junction remains open at asubstantially later point (see middle panel of FIG. 14B and C), and onlymuch later does it efficiently close (right hand panel of FIG. 14B andC). This suggests that different proteins translocate to the ER lumenvia strikingly different ribosome-membrane junctions. If, as suggestedby the studies on prion protein (PrP) biogenesis, signal sequences areinvolved in regulation of protein folding, this could occur in partthrough establishing different environments in which the nascent chainstarts to fold. When the ribosome-membrane junction remains open, as inthe middle panel of FIGS. 14B and 14C, the chain starts to fold in thereducing environment of the cytosol; when the ribosome-membrane junctionis closed, as in the middle pane of FIG. 14A and the right hand panelsof FIGS. 14B and 14C, the unfolded or partly folded chain proceeds tothe oxidizing environment of the ER lumen to initiate or continue theprocess of folding funnel selection.

As shown above (Example 1) in the case of the biogenesis of prionprotein (PrP), the open and closed states of the ribosome membranejunction correlate to the synthesis of secretory versus Ctm forms ofPrP, identical polypeptide sequences that are folded differently and indifferent transmembrane topological forms. The data in FIG. 14 extendsthis correlation of the state of the ribosome-membrane junction with thefolding of the protein, to simple secretory proteins such as prolactinand immunoglobulin heavy chain.

If signal sequences play the hypothesized regulatory role in proteinfolding, in part through the nature of the ribosome-membrane junctionthey establish, then swapping the signal sequence of two differentsecretory proteins should have a dramatic consequence, in the form ofthree predictions:

First, swapping of the signal sequence should change the environment inwhich the nascent chain resides (e.g.as assessed either by a change inthe ribosome-membrane junction as probed with proteinase K, or by achange in the proteins with which the nascent chain is associated asdetermined by chemical crosslinking). An example of a chemical crosslinkdifference between nascent chains of prolactin behind the prolactinsignal sequence and prolactin behind the IgG signal sequence is shown inFIG. 15. Substantailly the same conclusion was reached for PrP.

Second, the folding of the two proteins, directed down two differentfolding funnels by virtue of interaction with different accessoryproteins in different environments, is likely to be different. One wayto score this would be by engineering in glycosylation sites asreporters of protein conformation and demonstrating that the same chainis glycosylated when it is behind one signal sequence but not another,even though both signal sequences are ultimately cleaved from theprotein. This is demonstrated in FIG. 16 for prolactin with anengineered glycosylation reporter, behind either its own signal sequence(Prl) or the signal sequence of immunoglobulin heavy chain (IgG/Prl),growth hormone (GH/Prl). As can be seen from the 5^(th) lane of eachpanel in FIG. 16A, the resulting mature prolactin chain made behinddifferent signal sequences is conformationally different as assessed bythe ability of oligosaccharyl transferase to add core N-linked sugars tothe nascent chains. FIG. 16B quantitates this difference. Expression oftruncations of these constructs reveal that the difference inglycosylation of one prolactin compared to another, is not a kineticphenomenon because prolonged incubation of the truncated and thereforenon-growing but nascent chains, did not result in any further degree ofglycosylation. FIG. 16C shows that this difference in conformation, asscored by glycosylation, is readily apparent from material secreted intothe medium of cos cells transfected with the corresponding constructsencoding prolactin behind the various signal sequences. Thus theconformation of prolactin is changed simply by virtue of its synthesisbehind a different signal sequence. That difference cannot be accountedfor by the known roles of the signal sequence, because all signalsequences utilized are equally competent for targeting and translocationof both their native substates (see FIG. 13) and that encoded by themodified prolactin coding region. Furthermore, all conformations ofprolactin synthesized in FIG. 16 are judged as properly folded by thequality control machinery, as assessed by their secretion into themedium.

A third prediction is that, in some cases of swapped signal sequences,the “mis-match” between the signal and the subsequent passenger may besufficiently severe that even fully targeted chains are incapable offinding a folding funnel compatible with translocation into the ER lumenand may instead “fall back” into the cytosol, unable to be translocated.Precisely this form of defect was observed for a PrP mutant that couldonly be expressed in the secretory form with intact ER membranes, andwhose expression in glycoprotein-depleted reconstituted microsomalmembranes resulted in an inability of the chain to be translocated(Hegde et al, (1998) Mol Cel 2:85-91).

In FIG. 13 we demonstrate that behind the βlac and IgG signal sequences,prolactin displays a substantial translocation defect (FIG. 13A), whichwhich can be remedied by introduction of sequences from the region ofIgG subsequent to the signal sequence (FIG. 13B and 13C). Furthermorethe defect in mutant PrP translocation across glycoprotein-depletedreconstituted membranes can be corrected by swapping the PrP signalsequence for that of prolactin (data not shown), suggesting that thedefect was neither intrinsic to the membranes nor the chain, but rather,reflected an incompatibility of a particular signal sequence-chaincombination, consistent with the hypothesized role of the signalsequence in folding funnel selection.

Example 4

From the above, it can be seen that, the signal sequence can determinethe final folded state of the protein, as demonstrated by accessibilityof a glycosylation site engineered into the protein behind some signalsequences but not others. As the growing chain proceeds down differentfolding funnels, depending on the nature of the ribosome-membranejunction and protein-protein interactions between the nascent chain andthe translocation machinery, different conformations are achieved. As aresult of those differences in conformation, even while the chain isgrowing, the differently folded forms of a protein are differentiallyaccessible to modification enzymes. Hence, an engineered glycosylationsite serving as a reporter of protein conformation is glycosylated whenengineered behind one signal sequence but not another. Furthermore, inthe extreme case where the nature of the ribosome-membrane junction issuch as to allow substantial chain folding to be initiated prior totranslocation, additional information in the authentic chain must emergeto close the ribosome-membrane junction. Without such additionalinformation, the chain may fail to translocate, as demonstrated forprolactin behind the IgG signal sequence.

Thus, a model secretory protein recapitulates the observations madepreviously for the prion protein (Example 1), indicating that thoseresults were not a special case. Indeed, what appears to be specialabout the prion protein is not the pathway of its biogenesis andfolding, but that the alternative folding pathways manifest astopological differences, making them far more easy to detect.

Example 5 Modulation of Protein Folding by Trans Acting Factors

As shown in Example 1, above, the time course of PrP^(Sc) accrual intransmissible prion disease is followed closely by increased generationof ^(Ctm)PrP. Thus, the accumulation of PrP^(Sc) appears to modulate inTrans the events involved in generating or metabolising ^(Ctm)PrP.^(Ctm)PrP, which has its C terminal domain translocated to the ER lumen,triggers spontaneous neurodegeneration when overexpressed (Hegde,Science (1998) 279:827-834). As shown in Example 2, ^(Ctm)PrP appears tobe induced just prior to onset of clinical signs, suggesting that itinitiates a final common pathway to neurodegeneration. An as yet unknownglycoportein of the ER membrane is implicated as a translocationaccessory factor (TrAF) that “protects” the normal brain from expressionof ^(Ctm)PrP by directing nascent PrP chains to the pathway leading to^(sec)PrP (Hegde Mol Cell (1998) 2:85-89).

The mechanism by which nascent PrP chains are allocated among thetopological forms is complex (Rutkowski, submitted). The PrP signalsequence itself may play a role by establishing at least two populationsof nascent chains: some with an open ribosome-membrane junction; otherswith a closed ribosome-membrane junction. Thus, the signal sequence candetermine the environment faced by the emerging N-terminal domain ofPrP. Only the chains exposed to the cytosolic environment have thepotential to beco me ^(Ctm)PrP. While necessary, an openribosome-membrane junction is not sufficient to make ^(Ctm)PrP, asadditional protein-protein interactions determine the final outcome.Mutations that prevent the transmembrane domain from directing.^(Ctm)PrP formation result in these chains being redirected to thecytosol, where they most likely are degraded.

The distinction between ^(sec)PrP and ^(Ctm)PrP usually are made ontopological grounds. However, these two polypeptides of identicalsequence also differ in their conformation, as determined by theirdifferential sensitivity to limited protease digestion in non-denaturingdetergent solutions. Thus, translocational regulation appears to be ameans of generating multiple forms of PrP that differ in bothconfomation and function. The machinery (i.e. a TrAF) that directsnascent PrP chains to make ^(sec)PrP rather than ^(Ctm)PrP, may itselfbe regulated, based on the ability of scrapie infection to increase theamount of ^(Ctm)PrP detectable in brain. Together, these observationslead to a new principle: a protein's conformation is determined not justby its primary amino acid sequence, but also by proteins such as TrAF,that influence which of two or more different functional conformationaloutcomes actually occur or predominate.

Complex secretory or integral membrane proteins therefore can have manypotential functional folded states. The different folded states becomemanifest by a combinatorial series of interactions between ligandswithin the nascent chain and receptors at the translocon, in an array ofpossible environments (e.g. cytosol, membrane, lumen). The rates ofgeneration versus degradation of the various conformations determinewhich, and how many, of the possible final outcomes for a particularsubstrate are expressed at any given time. These processes areintegrated by signaling pathways from the cell surface and elsewhere,which coordinate protein biogenesis with both the specific immediateneeds (Chapman Annu Rev Cell and Dev Biol (1998) 14:459-485) and theglobal program of the cell (Bonafacino Nature (1990) 334:247-251)

This view necessitates that secretory and integral membrane nascentchains proceed down more than one folding funnel (or take differentpaths to different ends within a single, complex folding funnel). Thisis accomplished through the interaction of discrete sequences in thenascent chain with TrAFs or the environments that TrAFs make accessible.Different TrAFs, localized to different compartments (cytosol, membrane,ER lumen), that can act on different chains or different subsets offolding states of a given chain also contributes to the selection of onefunctional folded form versus another. This can be thought of as“third-order” complexity in protein folding. It differs from the conceptof molecular chaperones in that each of the possible structural outcomesare functional—just different in some aspect of function or regulation.Thus secretory and integral membrane proteins could exist in multipleconfomations, each with a distinctive functional signature.

Finally, the translocon and its components including TrAFs, that dictatethe choice among possible alternative folding funnels could themselvesbe subject to regulation by intracellular signaling, providing a “fourthorder” of complexity to protein folding. FIG. 18 summarizes a workingmodel of translocational regulation and relates it to the four orders ofprotein folding.

In order to explore translocational regulation in trans we havedeveloped fractionated and reconstituted systems in which, as a resultof manipulation of either the cytosol or the membranes, theconformational mix of PrP can be altered. Since the findings with PrPhave been shown (Rutkowski and Lingappa) to be reproduced by studies inother unrelated proteins (Rutkowski and Lingappa; Lingappa, Buhman, andFarese) it is clear that these systems will be amenable to manipulationin order to achieve the goals of detecting and studying the conformersof any signal sequence containing gene product of interest.

Results

Previously, the evidence for trans-acting influences affectingtranslocation and conformation have been relatively indirect (Hegde etal. (1998) Mol Cell 2:85-91; Hegde et al. (1999) Nat. 402:822-826). Wehave now found two new lines of evidence that support this notion moredirectly. First we have observed that cytosolic extracts prepared frommouse erythroleukemia (MEL) cells before and after differenciation areable to affect translocation of PrP, shifting the distribution of formsin favor of Sec-PrP (see FIG. 19). Second, when microsomal membraneswere prepared from hamster brain at various times in early post-nataldevelopment, TrAF activity was found to vary, going from high in day 13embryonic brain, to a nadir at post-natal day 14, in a manner consistentwith the hypothesis that TrAF activity, which suppresses Ctm-PrP, isdecreased in selected brain regions to promote Ctm-PrP and triggerneuronal apoptosis as part of the program of early rodent braindevelopment (see FIG. 20).

In view of the results described above, we set about to fractionate therabbit reticulocyte lysate in a manner that would be amenable tocomplementation with trans-acting factors from other sources. We foundthat a combination of ribosome pellet and DEAE eluate fraction weresufficient to restore full translational activity (see FIG. 21-23). Thefractionation methodology is described above.

Similarly, we modified previously published protocols (Gorlich andRapoport (1994) Cell 75:615-630; Hegde et al. (1998) Mol Cell 2:85-91)to generate fractionated and reconstituted proteoliposomes that werefunctionally equivalent to previous preparations, but which hadadvantages in terms of cost, speed, and amenability to furtherfractionation. This protocol is described above.

It is evident from the above results that the subject invention providesa new platform and paradigm for detecting, magnifying, understanding andmanipulating the participation of the translocation system inphysiological processes and the effect of different conformers of anysecretory or integral membrane protein, or other protein with a signalsequence for translocation across the ER membrane, on the physiology ofa host. By virtue of being able to control the folding of a protein,where different conformers may lead to different physiological outcomes,the opportunity to investigate the effect of the conformers on cellularpathways, the identification of agents involved with the formation ofthe different conformers, treatment for adverse results from aparticular conformation and understanding of the cellular processesresulting from the formation of the different conformers, can beachieved. New drugs can be developed and diseases can be considered fromdifferent viewpoints than have been heretofore employed.

The above results also demonstrate that microsomal membranes preparedfrom different species with different genetic composition provide apowerful tool for the expressing normally minor conformers. In thiscase, sea urchin microsomal membranes were found to be highlytranslocation competent, but to generate only one conformer of PrP,Ctm-PrP, a form that is normally a minor species in the presence ofmammalian microsomal membranes. The potential advantage of this approachover the biochemical approach is that labile components are less likelyto have been inactivated by the procedure, and multiple components canbe distinguished by the presence of distinctive partial activities indifferent membranes. Also the preparation of membranes from tissues ofprimitive organisms may be more cost-effective, at least for an initialscreen, with positive results confirmed with the more expensive,biochemically pure reagents.

The novel protocol provided for the preparation of fractionated andreconstituted proteoliposomes makes use of relatively inexpensivedetergents compatible with ion exchange chromatograpy, allowing forfurther fractionationof activities initially identified in the conA-depleted reconstitution, as well as activities whose initialidentification requires further fractionation of Con A flow-through. Afurther novel advantage oft he procedure provided here is that it makesthe initial screen for novel TrAFs prior to commitment of a majoreffort, quite non-labor intensive. This is especially important giventhe possibility that some TrAF activities may involve more than onecomponent and that, in some cases, one component in excess may be ableto partially compensate for lack of another component.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. All referencescited herein are incorporated herein by reference, as if set forth intheir entirety.

1.-13. (canceled)
 14. A method for detecting a conformational differencebetween proteins, said method comprising: analyzing a non-denaturedprotein by high pressure liquid chromatography, wherein said protein isobtained by expression of a chimeric gene having a DNA sequence encodingat least substantially the same amino acid sequence as an open readingframe encoding a native protein, wherein said native protein is encodedby a gene having a native signal sequence and said chimeric gene has asignal sequence other than said native signal sequence so that achimeric gene product is produced, wherein an alteration in thechromatographic profile of said chimeric gene product as compared to anative protein is indicative of a conformational difference between saidchimeric gene product and said native protein.
 15. The method accordingto claim 14, wherein said high pressure liquid chromatography isperformed using hydrophobic adsorption columns. 16.-55. (canceled)